Detection of Intracellular Reduced (Catalytically Active) SHP-1 and Analyses of Catalytically Inactive SHP-1 after Oxidation by Pervanadate or H2O2
细胞内还原的(具有催化活性的)SHP-1检测以及在经钒酸盐或H2O2氧化后无催化活性SHP-1的分析   

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Nature Immunology
Mar 2017

 

Abstract

Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends upon a ‘signature’ cysteine residue within the catalytic domain that is maintained in the de-protonated state at physiological pH rendering it susceptible to ROS-mediated oxidation. Direct and indirect techniques for detection of PTP oxidation have been developed (Karisch and Neel, 2013). To detect catalytically active PTPs, cell lysates are treated with iodoacetyl-polyethylene glycol-biotin (IAP-biotin), which irreversibly binds to reduced (S-) cysteine thiols. Irreversible oxidation of SHP-1 after treatment of cells with pervanadate or H2O2 is detected with antibodies specific for the sulfonic acid (SO3H) form of the conserved active site cysteine of PTPs. In this protocol, we describe a method for the detection of the reduced (S-; active) or irreversibly oxidized (SO3H; inactive) form of the hematopoietic PTP SHP-1 in thymocytes, although this method is applicable to any cysteine-dependent PTP in any cell type.

Keywords: Reactive oxygen species (活性氧种类), Protein tyrosine phosphatase (蛋白酪氨酸磷酸酶), Catalytic activity (催化活性)

Background

Reactive oxygen species (ROS) are generated by cellular NADPH oxidases and mitochondria. Most Protein Tyrosine Phosphatases (PTPs) contain a conserved catalytic cysteine with a low dissociation constant (pKa) that is highly susceptible to oxidation by ROS (Rudyk and Eaton, 2014). ROS-inactivation of PTPs plays an important role in regulating tyrosine-kinase-mediated signaling responses in numerous cell types. PTPs are rapidly oxidized in cells treated with the ROS H2O2 or the PTP inhibitor pervanadate (Huyer et al., 1997; Choi et al., 2017). Iodoacetyl-polyethylene glycol-biotin (IAP-biotin) selectively and irreversibly reacts with de-protonated (S-) cysteine thiols (Rudyk and Eaton, 2014). To label basal reduced (active) cellular PTPs, IAP-biotin is added at the time of cell lysis.

PTP active site cysteines can be oxidized by ROS to the sulfenic acid form (-SOH), which can then be converted into either sulfenylamide (-SN-) or cysteine disulfide (S-S) forms. Sulfenic acid, sulfenylamide and disulfide oxidized PTPs can be ‘re-activated’ by treatment with thiol reducing agents such as dithiothreitol (DTT) which convert the catalytic cysteine to the active (SH) state. PTP oxidation can be detected indirectly by a three-step method (irreversible alkylation of reduced active site cysteines with an alkylating agent followed by reduction of reversibly oxidized (SOH) active site cysteines with a reducing agent, then finally labeling of the newly formed cysteine thiols) (Weibrecht et al., 2007). This method for direct detection of reversibly oxidized PTPs is not widely used due to the fact that the sulfenic acid state is labile and transient (Karisch and Neel, 2013). PTP catalytic cysteines can also be progressively irreversibly ‘hyper-oxidized’ to the sulfinic (-SO2H) followed by the sulfonic (-SO3H) forms by higher concentrations of ROS or prolonged exposure to ROS. The availability of monoclonal antibodies specific for the sulfonic acid (-SO3H) form of the conserved active site of PTPs has provided a straightforward way to detect oxidized PTPs following treatment of cells with oxidizing agents (Persson et al., 2004). The susceptibility of PTPs in different cell populations to oxidation can be assessed by treatment of cells with oxidizing agents (varying the concentration of oxidizing agent and/or treatment time) followed by blotting of proteins from cell lysates after separation by SDS-PAGE with anti-oxidized-PTP mAb. Here, we describe methods used to detect either reduced (S-; active) or irreversibly oxidized (SO3H; inactive) SHP-1 in thymocytes.

Materials and Reagents

  1. Consumables
    1. Push pins or syringe needles
    2. Pipette tips (Neptune Scientific, catalog numbers: BT10F , BT100 , BT1250 )
    3. 14 ml conical tubes (Corning, Falcon®, catalog number: 352059 )
    4. 1.6 ml microfuge tubes (Neptune Scientific, catalog number: 4445.S.X )
    5. 3MM paper (GE Healthcare, catalog number: 3030-861 )
    6. 6-well plate (Nest Biotechnology, catalog number: 703001 )
    7. Cell strainer, 70 μm (Southern Labware, catalog number: C4070 )
    8. PVDF membrane (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88518 )
    9. Western blotting film (Next Day Science, catalog number: A8803 )
    10. Nitrocellulose membrane (GE Healthcare, catalog number: 10600002 )

  2. Biological reagents
    1. Female 6-8 week old C57BL/6 mouse (Taconic Biosciences, catalog number: B6NTac )

  3. Chemical reagents
    1. Trypan blue solution, 0.4% (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
    2. Serum-free medium (Corning, Mediatech, catalog number: 40-101-CV )
    3. Methanol (Sigma-Aldrich, catalog number: 32213 )
    4. 1 M HEPES pH 7.0 (Sigma-Aldrich, catalog number: H0887 )
    5. 1 M Tris-HCl pH 7.5 (Quality Biological, catalog number: 351-006-101 )
    6. 5 M NaCl (Quality Biological, catalog number: 351-036-101 )
    7. 10% SDS (Quality Biological, catalog number: 351-032-101 )
    8. Sodium deoxycholate (Sigma-Aldrich, catalog number: D6750 )
    9. Nonidet P40 (Sigma-Aldrich, Roche Diagnostics, catalog number: 11754599001 )
    10. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
    11. Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S7920 )
    12. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
    13. Iodoacetyl PEG-biotin (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 21334 )
    14. Diethylenetriaminepentaacetic acid (DTPA) (Sigma-Aldrich, catalog number: D6518 )
    15. Catalase (Sigma-Aldrich, catalog number: C30 )
    16. Protease inhibitor tablets (Roche Diagnostics, catalog number: 5056489001 )
    17. Anti-Oxidized PTP active site mAb (R&D Systems, catalog number: MAB2844 )
    18. Anti-SHP-1 antibody (Santa Cruz Biotechnology, catalog number: sc-287 )
    19. Goat anti-mouse IgG HRP (Santa Cruz Biotechnology, catalog number: sc-2302 )
    20. Mouse anti-rabbit IgG HRP (Santa Cruz Biotechnology, catalog number: sc-2357 )
    21. GammaBind G Sepharose (GE Healthcare, catalog number: 17088501 )
    22. SDS protein gel loading solution (Quality Biological, catalog number: 351-082-661 )
    23. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
    24. Tris-glycine transfer buffer (25x) (Thermo Fisher Scientific, InvitrogenTM, catalog number: LC3675 )
    25. Tris-glycine SDS running buffer (10x) (Thermo Fisher Scientific, InvitrogenTM, catalog number: LC2675 )
    26. NovexTM Wedge well 10% Tris-glycine gel (Thermo Fisher Scientific, InvitrogenTM, catalog number: XP00100 )
    27. Non-fat dry milk (Santa Cruz Biotechnology, catalog number: sc-2325 )
    28. Streptavidin-HRP (GE Healthcare, catalog number: RPN1231-2ML )
    29. SuperSignalTM West Pico chemiluminescent substrate (Thermo Fisher Scientific, catalog number: 34080 )
    30. Tween 20 (Sigma-Aldrich, catalog number: P7949 )
    31. EGTA (Sigma-Aldrich, catalog number: 03777 )
    32. β-Glycerophosphate (Sigma-Aldrich, catalog number: G6501 )
    33. N-Ethylmaleimide (Sigma-Aldrich, catalog number: E3876 )
    34. H2O2 (3%) (Sigma-Aldrich, catalog number: 88597 )
    35. Sodium orthovanadate (Na3VO4) (Sigma-Aldrich, catalog number: 450243 )
    36. Iodoacetamide (Sigma-Aldrich, catalog number: I1149 )
    37. Oxidation lysis buffer (see Recipe 1)
    38. Oxidation wash buffer (see Recipe 2)
    39. TBST buffer (see Recipe 3)
    40. 1 mM pervanadate (see Recipe 4)
    41. Lysis buffer (see Recipe 5)

Equipment

  1. VANTAGE Strabismus Scissors (Steeles, model: V95-312 )
  2. Forceps (Polysciences, model: 5 Dumont INOX )
  3. Pipettes (Gilson, models: P10, P100, P1000; catalog numbers: F144802 , F123615 , F123602 )
  4. Clay Adams Nutator mixer (BD, model: 421105 )
  5. Mini tank transfer unit (GE Healthcare, catalog number: TE22 )
  6. X cell SureLockTM mini-cell (Thermo Fisher Scientific, InvitrogenTM, catalog number: EI0001 )
  7. Hemocytometer (Daigger Scientific, catalog number: EF16034F )
  8. CO2 incubator (Thermo Fisher Scientific, model: HeracellTM 150 )
  9. Platform shaker (Heidolph, model: Unimax 1010 , catalog number: 036130180)
  10. Centrifuge (Eppendorf, model: 5418 , catalog number: 5401000137)
  11. Chemical resistant vacuum pump (Southern Labware, model: Model 400 )
  12. Table top centrifuge for 14 ml tubes (Thermo Fisher Scientific, model: SorvallTM LegendTM XTR )
  13. Film developer (Kodak, model: KODAK X-OMAT 2000 Processor )

Software

  1. MultiGauge (Fujifilm software)
  2. GraphPad Prism (GraphPad software)

Procedure

  1. Isolation of thymocytes
    1. Euthanize mouse following approved IACUC protocol.
    2. Isolate thymus from mice using scissors and forceps (see Figure 1).


      Figure 1. Removal of thymus from a euthanized mouse. Secure mouse limbs to cutting board with push pins or syringe needles. Carefully cut skin with scissors down the midline from the neck to the pelvis. Make short lateral cuts in the skin at the top and bottom of the midline cut and secure skin flaps to the cutting board. A. Grab xyphoid (yellow arrow) with forceps and gently lift, then cut sternum down the midline with scissors and fold back rib cage to expose thymus (B, blue arrow). Gently lift thymus with forceps in one hand and with forceps in other hand dislodge thymus from surrounding tissues by gentle teasing. C. Thymus (note 2 lobes) after removal from the mediastinum.

    3. Transfer thymus to one well of a 6-well plate filled with 1 ml of degassed serum-free media (see Note 1) then cut thymus into several small sections with scissors.
    4. Gently compress thymus pieces in a cell strainer with forceps to release thymocytes from stroma.
    5. Add 9 ml of degassed serum-free media on ice.
    6. Filter the thymocyte suspension by pipetting (1,000 μl pipette) through a cell strainer into a 14 ml tube.
    7. Centrifuge for 1 min at 1,000 x g, 4 °C.
    8. Remove the supernatant and resuspend the pellet with 10 ml of degassed serum-free media on ice.

  2. Detection of reduced (catalytically active) SHP-1
    1. Enumerate thymocytes using a hemocytometer and trypan blue solution. The expected number of cells per thymus is 0.5-1 x 108.
    2. Put 1 x 107 cells into a 14 ml conical tube and centrifuge for 1 min at 1,000 x g, 4 °C.
    3. Discard supernatant and resuspend the pellet in 0.5 ml of degassed oxidation lysis buffer (Recipe 1, Note 1) on ice. Transfer to a 1.6 ml microfuge tube. Degassed oxidation lysis buffer without IAP-biotin is used as a control.
    4. Vortex for 15 sec.
    5. Incubate the lysate for 20 min on ice.
    6. Centrifuge for 10 min at 12,000 x g, 4 °C.
    7. Transfer supernatant to a 1.6 ml microfuge tube and add 0.5 ml of degassed oxidation lysis buffer.
    8. Add 5 μl (1 μg) of anti-SHP-1 Ab and incubate overnight in a nutator at 4 °C.
    9. Add 20 μl of degassed oxidation wash buffer (Recipe 2) equilibrated GammaBind G-Sepharose and incubate in a nutator for 1 h at 4 °C.
    10. Add 1 ml of degassed oxidation buffer, pipet to disperse sepharose then centrifuge for 1 min at 1,000 x g. Remove buffer by aspiration. Repeat three times.
    11. Add 30 μl of SDS protein gel loading solution containing 5% 2-mercaptoethanol to pellet.
    12. Boil samples for 5 min and load onto one lane of 10% Tris-glycine SDS-PAGE gel.
    13. Run the gel for 3 h at 100 V then transfer to PVDF membrane by electro-blotting.
      Note: PVDF membrane is soaked in 10 ml of methanol for 5 min and then in 15 ml of transfer buffer for 5 min before electro-blot transfer.
    14. Block PVDF membrane with 5% non-fat dry milk in TBST buffer (Recipe 3) for 1 h at room temperature on a platform shaker.
    15. Add Streptavidin-HRP at a 1:5,000 dilution in a 5 ml of TBST buffer for 4 h at room temperature on a platform shaker.
    16. Wash 3 x 10 min in 15 ml of TBST buffer at room temperature.
    17. Add 2 ml of SuperSignalTM West Pico chemiluminescent developing solution and incubate for 5 min.
    18. Pour off the developing solution.
    19. In a darkroom, place X-ray films on the wrapped membrane.
    20. Expose for 1 min (see Note 2). Develop the film.

  3. Analyses of SHP-1 oxidation after stimulation with pervanadate or H2O2.
    1. Enumerate thymocytes as described above.
    2. Put 1 x 107 cells into a 14 ml conical tube and centrifuge for 1 min at 1,000 x g, 4 °C.
    3. Discard supernatant, add 0.1 ml of degassed serum-free media and gently resuspend cells by repeated pipetting.
    4. Add 0.1 ml of 2x pervanadate (Recipe 4) for 10 min or 0.1 ml 2x H2O2 (2-20 mM) for 5 min at room temperature.
      Note: 3% (w/v) H2O2 is 880 mM. Optimal final concentrations of H2O2 and pervanadate will need to be established for each cell type and will depend on the extent of PTP oxidation desired. Initial experiments should test a range of concentrations. Suggested ranges: Pervanadate, 10-200 μM (final concentration); H2O2: 0.1-10 mM (final concentration). Cells are also treated with the same amount of vehicle solution without H2O2 or pervanadate as a negative control.
    5. Stop by centrifugation for 1 min at 1,000 x g at room temperature.
    6. Discard supernatant and wash the pellet once with degassed 1x PBS.
    7. Resuspend with 0.5 ml of degassed lysis buffer (Recipe 5).
    8. Incubate the lysates for 20 min on ice.
    9. Centrifuge for 10 min at 12,000 x g, 4 °C.
    10. Take supernatant, transfer to a 1.6 ml microfuge tube and add 0.5 ml of degassed lysis buffer.
    11. Add 5 μl (1 μg) of anti-SHP-1 Ab and incubate in a nutator overnight at 4 °C.
    12. Add 20 μl of degassed lysis buffer-equilibrated GammaBind G-Sepharose and incubate in a nutator for 1 h at 4 °C.
    13. Wash by pelleting (centrifuge for 1min at 1,000 x g), aspiration, and resuspension with 1 ml of degassed lysis buffer three times.
    14. Add 30 μl of SDS protein gel loading solution containing 5% 2-mercaptoethanol to pellet.
    15. Boil samples for 5 min and load each sample into well of 10% Tris-glycine SDS-PAGE gel.
    16. Run the gel for 3 h at 100 V and transfer to nitrocellulose membrane by electroblotting.
    17. Block nitrocellulose membrane with 10 ml of 5% non-fat dry milk in TBST buffer for 1 h at room temperature on a platform shaker.
    18. Incubate with anti-oxidized PTP active site Ab in 5 ml of TBST buffer for 4 h at room temperature.
      Pour off the antibody solution.
      Note: Anti-oxidized PTP active site antibody detects the conserved PTP active site with the catalytic cysteine residue oxidized to a sulfonic acid form (Persson et al., 2004).
    19. Wash twice with 10 ml TBST at room temperature.
    20. Add Goat anti-mouse IgG HRP at a 1:2,000 dilution in a 5 ml of TBST buffer for 1 h at room temperature.
    21. Pour off secondary antibody solution.
    22. Wash 3 x 10 min with 15 ml of TBST buffer at room temperature.
    23. Add 2 ml of SuperSignalTM West Pico chemiluminescent developing solution and incubate for 5 min.
    24. Pour off the developing solution.
    25. In a darkroom, put X-ray films on the wrapped membrane.
    26. Expose for 1 min (see Note 2). Develop the film.
    27. See Figure 2 and Figure 3 for representative results.


      Figure 2. Analysis of reduced (active) SHP-1 in thymocytes. Immunoblot analysis of anti-SHP-1 immunoprecipitates from total thymocytes from wild type (Themis+/+; WT) or Themis-/- (KO) mice after labeling reduced active PTP active site cysteines with iodoacetyl-polyethylene glycol-biotin. The blot was probed with streptavidin-horseradish peroxidase (SA-HRP) to detect biotinylated proteins. The reduced SHP-1 was quantitated by densitometry. Values are normalized to total SHP-1 band density in each experiment.


      Figure 3. SHP-1 oxidation after stimulation with pervanadate or H2O2 treatment. Immunoblot analysis of active-site oxidation of SHP-1 in total thymocytes treated with various concentrations of pervanadate (PV) (A) or H2O2 (B). Proteins immunoprecipitated with anti-SHP1 were analyzed, and the blots were probed with antibody specific for the sulfonic acid (SO3H) form of the conserved active site cysteine of PTPs for the detection of irreversible oxidation of SHP1.

Data analysis

For quantitation of band density, we used MultiGauge (Fujifilm software). At least three, preferably 4-6 independent experiments were performed for statistical analysis. For statistical analysis, we used GraphPad Prism (GraphPad software). Typically, we provide a representative blot that was used for statistical analysis in the body of the paper and provide all additional blots used for statistical analysis in the supplemental section. Since band densities are normalized to control bands (e.g., total SHP-1 blots), all data should be usable and included unless there were technical problems with a particular gel or blot. Normalization to control total protein bands is considered essential especially if separate blots are included in the statistical analysis. We have also found that the marginal lanes on gels (first and last) can be problematic, so we try to avoid using those two lanes for critical samples. See Choi et al. (2017) for an example of published data from these experiments.

Notes

  1. All solutions and media should be degassed for 1 h with a membrane vacuum pump (37 L/min) and stored on ice.
  2. Exposure time will be different for each antibody.

Recipes

  1. Oxidation lysis buffer
    50 mM Tris (pH 7.5)
    100 mM NaCl
    0.1% SDS
    0.5% sodium deoxycholate
    0.5% Nonidet P-40
    0.5% Triton X-100
    50 mM NaF
    1 mM PMSF
    0.4 mM Iodoacetyl PEG-biotin
    100 μM DTPA
    200 U/ml catalase
    1 tablet of protease inhibitor per 50 ml
  2. Oxidation wash buffer
    50 mM Tris (pH 7.5)
    100 mM NaCl
    0.5% Nonidet P-40
    0.5% Triton X-100
    50 mM NaF
  3. TBST buffer
    20 mM Tris (pH 7.5)
    135 mM NaCl
    0.05% Tween 20
  4. 1 mM pervanadate
    1 mM Na3VO4
    5 mM H2O2
    Note: Make fresh and incubate for 5 min at room temperature before use.
  5. Lysis buffer
    1% Nonidet P-40
    10 mM Tris (pH 7.5)
    150 mM NaCl
    2 mM EGTA
    50 mM β-glycerophosphate
    2 mM Na3VO4
    10 mM NaF
    10 mM Iodoacetamide
    10 mM NEM
    1 tablet of protease inhibitor per 50 ml

Acknowledgments

This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver, National Institute of Child Health and Human Development (1ZIAHD001803 to P.E.L.). The protocol has been adapted from Choi et al. (2017) Nature Immunology 18, 433-441. The authors have no conflicts of interests or competing interests relating to this publication.

References

  1. Choi, S., Warzecha, C., Zvezdova, E., Lee, J., Argenty, J., Lesourne, R., Aravind, L. and Love, P. E. (2017). THEMIS enhances TCR signaling and enables positive selection by selective inhibition of the phosphatase SHP-1. Nat Immunol 18(4): 433-441.
  2. Huyer, G., Liu, S., Kelly, J., Moffat, J., Payette, P., Kennedy, B., Tsaprailis, G., Gresser, M. J. and Ramachandran, C. (1997). Mechanism of inhibition of protein tyrosine phosphatases by vanadate and pervanadate. J Biol Chem 272: 843-51.
  3. Karisch, R. and Neel, B. G. (2013). Methods to monitor classical protein-tyrosine phosphatase oxidation. FEBS J 280(2): 459-475.
  4. Persson, C., Sjoblom, T., Groen, A., Kappert, K., Engstrom, U., Hellman, U., Heldin, C. H., den Hertog, J. and Ostman, A. (2004). Preferential oxidation of the second phosphatase domain of receptor-like PTP-α revealed by an antibody against oxidized protein tyrosine phosphatases. Proc Natl Acad Sci U S A 101(7): 1886-1891.
  5. Rudyk, O. and Eaton, P. (2014). Biochemical methods for monitoring protein thiol redox states in biological systems. Redox Biol 2: 803-813.
  6. Weibrecht, I., Bohmer, S. A., Dagnell, M., Kappert, K., Ostman, A. and Bohmer, F. D. (2007). Oxidation sensitivity of the catalytic cysteine of the protein-tyrosine phosphatases SHP-1 and SHP-2. Free Radic Biol Med 43(1): 100-110.

简介

细胞活性氧(ROS)对半胱氨酸依赖性蛋白酪氨酸磷酸酶(PTP)的氧化失活在调节多种细胞类型的信号转导中起关键作用。大多数PTP的磷酸酶活性取决于催化结构域内的“标记”半胱氨酸残基,其在生理pH下保持质子化状态,使其易受ROS介导的氧化。已经开发了用于检测PTP氧化的直接和间接技术(Karisch和Neel,2013)。为了检测催化活性的PTP,用碘乙酰 - 聚乙二醇 - 生物素(IAP-生物素)处理细胞裂解物,所述碘乙酰 - 聚乙二醇 - 生物素(IAP-生物素)不可逆地结合还原的(S-5)半胱氨酸硫醇。使用对磺酸(SO 3)特异性的抗体检测用过钒酸盐或H 2 O 2 2处理细胞后SHP-1的不可逆氧化, H)形式的PTP的保守的活性位点半胱氨酸。在该协议中,我们描述了用于检测造血PTP SHP的还原(S ; active)或不可逆氧化(SO 3 H;非活性)形式的方法-1,尽管这种方法适用于任何细胞类型中的任何半胱氨酸依赖性PTP。

【背景】活性氧(ROS)由细胞NADPH氧化酶和线粒体产生。大多数蛋白质酪氨酸磷酸酶(PTP)含有保守的催化半胱氨酸,其具有低的解离常数(pKa),其对ROS的氧化非常敏感(Rudyk和Eaton,2014)。 PTP的ROS失活在许多细胞类型中调节酪氨酸激酶介导的信号传导反应中起重要作用。在用ROS H 2 O 2或PTP抑制剂过钒酸盐处理的细胞中,PTP被快速氧化(Huyer等,1997; Choi等,等),2017)。碘代乙酰 - 聚乙二醇 - 生物素(IAP-生物素)选择性且不可逆地与去质子化(S-S) - 半胱氨酸硫醇反应(Rudyk和Eaton,2014)。为了标记基本还原(活性)细胞PTP,在细胞裂解时加入IAP-生物素。

PTP活性位点半胱氨酸可被ROS氧化为次磺酸形式(-SOH),然后可将其转化成磺酰胺(-SN-)或半胱氨酸二硫(S-S)形式。亚硫酸,次磺酰胺和二硫化物氧化的PTP可以通过用巯基还原剂例如将催化半胱氨酸转化为活性(SH)状态的二硫苏糖醇(DTT)处理来“再活化”。可以通过三步法间接检测PTP氧化(还原的活性部位半胱氨酸用烷化剂不可逆地烷基化,然后用还原剂还原可逆氧化的(SOH)活性部位半胱氨酸,最后标记新形成的半胱氨酸硫醇)(Weibrecht et al。,2007)。这种直接检测可逆氧化PTP的方法由于亚磺酸状态不稳定和短暂(Karisch和Neel,2013),因此没有被广泛使用。 PTP催化半胱氨酸也可以逐渐不可逆地“高度氧化”成亚磺酸(-SO 2 H),接着是磺酸(-SO 3 H)形式,通过较高浓度ROS或长时间暴露于ROS。对PTP保守活性位点的磺酸(-SO 3 H)形式特异性的单克隆抗体的可用性提供了直接的方式来检测用氧化剂处理细胞后的氧化PTP(Persson,等人,2004年)。通过用氧化剂处理细胞(改变氧化剂的浓度和/或处理时间),然后通过SDS-PAGE分离细胞裂解物中的蛋白质后,用抗-CD20抗体评估PTP在不同细胞群中的氧化的易感性,氧化-PTP mAb。在这里,我们描述了用于检测在胸腺细胞中减少的(S - ; active)或不可逆氧化的(SO 3 H;无活性)SHP-1的方法。

关键字:活性氧种类, 蛋白酪氨酸磷酸酶, 催化活性

材料和试剂

  1. 耗材
    1. 推针或注射器针
    2. 移液器吸头(Neptune Scientific,产品目录号:BT10F,BT100,BT1250)
    3. 14ml锥形管(Corning,Falcon ,目录号:352059)
    4. 1.6 ml离心管(Neptune Scientific,目录号:4445.S.)
    5. 3MM纸(GE Healthcare,目录号:3030-861)
    6. 6孔板(Nest Biotechnology,目录号:703001)
    7. 细胞过滤器,70微米(Southern Labware,目录号:C4070)
    8. PVDF膜(Thermo Fisher Scientific,Thermo Scientific TM,目录号:88518)
    9. 蛋白质印迹膜(Next Day Science,目录号:A8803)
    10. 硝酸纤维素膜(GE Healthcare,目录号:10600002)

  2. 生物试剂
    1. 雌性6-8周龄的C57BL / 6小鼠(Taconic Biosciences,目录号:B6NTac)

  3. 化学试剂
    1. 台盼蓝溶液,0.4%(Thermo Fisher Scientific,Gibco TM,产品目录号:15250061)
    2. 无血清培养基(Corning,Mediatech,目录号:40-101-CV)
    3. 甲醇(Sigma-Aldrich,目录号:32213)
    4. 1M HEPES pH7.0(Sigma-Aldrich,目录号:H0887)
    5. 1M Tris-HCl pH 7.5(Quality Biological,目录号:351-006-101)
    6. 5M NaCl(Quality Biological,目录号:351-036-101)
    7. 10%SDS(Quality Biological,目录号:351-032-101)
    8. 脱氧胆酸钠(Sigma-Aldrich,目录号:D6750)
    9. Nonidet P40(Sigma-Aldrich,Roche Diagnostics,目录号:11754599001)
    10. Triton X-100(Sigma-Aldrich,目录号:T8787)
    11. 氟化钠(NaF)(Sigma-Aldrich,目录号:S7920)
    12. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
    13. 碘乙酰PEG-生物素(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:21334)
    14. 二亚乙基三胺五乙酸(DTPA)(Sigma-Aldrich,目录号:D6518)
    15. 过氧化氢酶(Sigma-Aldrich,目录号:C30)
    16. 蛋白酶抑制剂片(Roche Diagnostics,目录号:5056489001)
    17. 抗氧化的PTP活性位点mAb(R& D Systems,目录号:MAB2844)
    18. 抗SHP-1抗体(Santa Cruz Biotechnology,目录号:sc-287)
    19. 山羊抗小鼠IgG HRP(Santa Cruz Biotechnology,目录号:sc-2302)
    20. 小鼠抗兔IgG HRP(Santa Cruz Biotechnology,目录号:sc-2357)
    21. GammaBind G Sepharose(GE Healthcare,目录号:17088501)
    22. SDS蛋白凝胶上样溶液(Quality Biological,目录号:351-082-661)
    23. 2-巯基乙醇(Sigma-Aldrich,目录号:M3148)
    24. Tris-甘氨酸转移缓冲液(25x)(Thermo Fisher Scientific,Invitrogen TM,目录号:LC3675)
    25. Tris-甘氨酸SDS运行缓冲液(10x)(Thermo Fisher Scientific,Invitrogen TM,目录号:LC2675)
    26. Novex TM楔形10%Tris-甘氨酸凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:XP00100)
    27. 脱脂奶粉(Santa Cruz Biotechnology,目录号:sc-2325)
    28. 链霉亲和素-HRP(GE Healthcare,目录号:RPN1231-2ML)
    29. SuperSignal TM West Pico化学发光底物(Thermo Fisher Scientific,目录号:34080)
    30. 吐温20(Sigma-Aldrich,目录号:P7949)
    31. EGTA(Sigma-Aldrich,目录号:03777)
    32. β-甘油磷酸酯(Sigma-Aldrich,目录号:G6501)
    33. N-乙基马来酰亚胺(Sigma-Aldrich,目录号:E3876)
    34. (2%)(Sigma-Aldrich,目录号:88597)。
    35. 原钒酸钠(Na 3 VO 4)(Sigma-Aldrich,目录号:450243)
    36. 碘乙酰胺(Sigma-Aldrich,目录号:I1149)
    37. 氧化裂解缓冲液(见方法1)
    38. 氧化清洗缓冲液(见配方2)
    39. TBST缓冲液(见方法3)
    40. 1 mM pervanadate(见配方4)
    41. 裂解缓冲液(见方法5)

设备

  1. VANTAGE斜视剪(Steeles,型号:V95-312)
  2. 镊子(Polysciences,型号:5 Dumont INOX)
  3. 移液器(Gilson,型号:P10,P100,P1000;目录号:F144802,F123615,F123602)
  4. 粘土亚当斯Nutator搅拌机(BD,型号:421105)
  5. 微型槽罐转运单元(GE Healthcare,目录号:TE22)
  6. X细胞SureLock TM迷你细胞(Thermo Fisher Scientific,Invitrogen TM,目录号:EI0001)。
  7. 血细胞计数器(Daigger Scientific,目录号:EF16034F)
  8. CO 2培养箱(Thermo Fisher Scientific,型号:Heracell TM 150)
  9. 平台摇床(Heidolph,型号:Unimax 1010,目录号:036130180)
  10. 离心机(Eppendorf,型号:5418,目录号:5401000137)
  11. 耐化学腐蚀真空泵(Southern Labware,型号:400型)
  12. 用于14ml管的台式离心机(Thermo Fisher Scientific,型号:Sorvall TM Legend TM XTR)
  13. 胶片开发商(柯达,型号:柯达X-OMAT 2000处理器)

软件

  1. MultiGauge(富士软件)
  2. GraphPad Prism(GraphPad软件)

程序

  1. 胸腺细胞的分离
    1. 根据批准的IACUC协议安乐死鼠标。

    2. 用剪刀和镊子从小鼠中分离胸腺(见图1)

      图1.去除安乐死小鼠的胸腺。 用推针或注射器针将鼠标四肢固定在切割板上。用剪刀从颈部到骨盆中线小心切开皮肤。在中线的顶部和底部做皮肤短侧切,并将皮瓣固定在砧板上。 A.用钳子抓住xyphoid(黄色箭头)并轻轻抬起,然后用剪刀剪下胸骨中线,折回胸廓以暴露胸腺(B,蓝色箭头)。一方面用镊子轻轻地提起胸腺,另一方面用镊子轻轻地取笑,从周围组织中取出胸腺。 C.从胸腔中摘除胸腺(注2)。

    3. 将胸腺转移到装有1ml脱气的无血清培养基的6孔板的一个孔中(见注1),然后用剪刀将胸腺切成几个小部分。

    4. 。用镊子轻轻压缩细胞过滤器中的胸腺碎片,从基质释放胸腺细胞。

    5. 加入9毫升脱气的无血清培养基
    6. 过滤胸腔细胞悬液(1000微升移液器)通过细胞过滤器到14毫升管。

    7. 在1000×g,4℃下离心1分钟
    8. 取出上清液并用10ml脱气的无血清培养基在冰上重悬沉淀。

  2. 还原(催化活性)SHP-1的检测
    1. 使用血细胞计数器和台盼蓝溶液来枚举胸腺细胞。每个胸腺细胞的预期数目是0.5-1×10 8。
    2. 将1×10 7个细胞放入14ml锥形管中并在1000℃,4℃下离心1分钟。
    3. 弃去上清,并在冰上重新悬浮在0.5ml脱气氧化裂解缓冲液(配方1,注1)中。转移到1.6毫升的微量离心管中。
      使用不含IAP-生物素的脱气氧化裂解缓冲液作为对照
    4. 涡旋15秒。

    5. 在冰上孵育裂解液20分钟

    6. 在12,000×g,4℃下离心10分钟
    7. 将上清液转移到1.6 ml微量离心管中,并加入0.5 ml脱气氧化裂解液。
    8. 添加5微升(1微克)抗SHP-1 Ab,并在4°C的nutator孵育过夜。
    9. 加入20μl脱气氧化洗涤缓冲液(配方2)平衡的GammaBind G-琼脂糖,并在4°C在nutator孵育1小时。
    10. 加入1ml脱气的氧化缓冲液,吸取分散琼脂糖凝胶,然后在1000gxg下离心1分钟。通过抽吸去除缓冲液。重复三次。

    11. 加入30μl含有5%2-巯基乙醇的SDS蛋白凝胶上样溶液
    12. 将样品煮沸5分钟并加载到10%Tris-甘氨酸SDS-PAGE凝胶的一条泳道上。

    13. 在100 V下运行凝胶3 h,然后通过电印迹转移到PVDF膜上 注意:PVDF膜在10ml甲醇中浸泡5分钟,然后在15ml转移缓冲液中浸泡5分钟,然后进行电转印。
    14. 在平台振荡器上,在室温下用TBST缓冲液(配方3)中的5%脱脂奶粉封闭PVDF膜1小时。

    15. 在室温下,在5 ml TBST缓冲液中以1:5,000稀释度加入链霉抗生物素蛋白-HRP 4小时

    16. 在15毫升的TBST缓冲液中室温下3×10分钟
    17. 加入2ml SuperSignal West Pico化学发光显影液并孵育5分钟。
    18. 倒掉开发中的解决方案。
    19. 在暗室中,将X光胶片放在包裹的膜上。
    20. 暴露1分钟(见注2)。开发电影。

  3. 用过钒酸盐或H 2 O 2 O 2刺激后SHP-1氧化的分析。
    1. 如上所述枚举胸腺细胞。
    2. 将1×10 7个细胞放入14ml锥形管中并在1000℃,4℃下离心1分钟。
    3. 弃去上清,加入0.1ml脱气的无血清培养基,反复吹打轻轻悬浮细胞。
    4. 在室温下加入0.1ml 2x过钒酸盐(方案4)10分钟或0.1ml 2x H 2 O 2(2-20mM)5分钟。 /> 注:3%(w / v)H 2 0 2 > 是880mM。 H的最佳终浓度 2 2 2 并且需要为每种细胞类型建立过钒酸盐,并将取决于所需的PTP氧化程度。最初的实验应该测试一系列的浓度。建议的范围:10-200μM的Pervanadate(终浓度); H 2 0 2 :0.1-10 mM(终浓度)。细胞也用相同数量的载体溶液处理而不用H 2 / em> 2 2 2 > 或者pervanadate作为阴性对照。

    5. 在室温下1000×g离心1分钟停止。
    6. 弃去上清,用脱气的1x PBS洗一次沉淀。
    7. 用0.5ml脱气的裂解缓冲液重悬(方案5)。

    8. 在冰上孵育裂解物20分钟

    9. 在12,000×g,4℃下离心10分钟
    10. 取上清液,转移到1.6 ml微量离心管中,加入0.5 ml脱气裂解液。
    11. 加入5微升(1微克)的抗SHP-1抗体,并在4摄氏度的nutator孵育过夜。
    12. 加入20μl脱气的裂解缓冲液平衡的GammaBind G-琼脂糖,并在4摄氏度在nutator中孵育1小时。
    13. 通过造粒洗涤(1000×g离心1分钟),抽吸,并用1ml脱气的裂解缓冲液重新悬浮三次。

    14. 加入30μl含有5%2-巯基乙醇的SDS蛋白凝胶上样溶液
    15. 将样品煮沸5分钟,并将每个样品加样到10%Tris-甘氨酸SDS-PAGE凝胶中。

    16. 在100 V下运行凝胶3 h,然后通过电印迹转移到硝酸纤维素膜上
    17. 在平台振荡器上,用硝酸纤维素膜和10毫升含5%脱脂奶粉的TBST缓冲液在室温下封闭1小时。
    18. 在室温下用5 ml TBST缓冲液与抗氧化PTP活性位点Ab孵育4 h。
      倒掉抗体溶液。
      注意:抗氧化的PTP活性位点抗体检测到保守的PTP活性位点,其催化的半胱氨酸残基被氧化成磺酸形式(Persson et al。,2004)。

    19. 在室温下用10毫升TBST洗两次
    20. 在室温下,在5ml TBST缓冲液中以1:2,000稀释度添加山羊抗小鼠IgG HRP 1小时。
    21. 倒掉二抗溶液。
    22. 在室温下用15毫升TBST缓冲液洗3次10分钟。
    23. 加入2 ml SuperSignal West Pico化学发光显影液,孵育5分钟。
    24. 倒掉开发中的解决方案。
    25. 在暗室里,把X光胶片放在包裹膜上。
    26. 暴露1分钟(见注2)。开发电影。

    27. 见图2和图3

      图2.胸腺细胞中还原(活性)SHP-1的分析。来自野生型(Themis + / + WT;或Themis( - / - )(KO)小鼠的总胸腺细胞的抗SHP-1免疫沉淀物的免疫印迹分析用碘乙酰 - 聚乙二醇 - 生物素标记减少的活性PTP活性位点半胱氨酸后。用链霉亲和素 - 辣根过氧化物酶(SA-HRP)探测印迹以检测生物素化蛋白质。通过密度测定法定量减少的SHP-1。
      在每个实验中,将数值标准化为总SHP-1条带密度

      图3.用过钒酸盐或H 2 O 2 2处理刺激后的SHP-1氧化 SHP-1的活性位点氧化的免疫印迹分析在用不同浓度的过钒酸盐(PV)(A)或H 2 O 2(B)处理的总胸腺细胞中。分析用抗SHP1免疫沉淀的蛋白质,并用对PTP的保守活性位点半胱氨酸的磺酸(SO 3 H)形式特异性的抗体探测印迹,以检测SHP1的不可逆氧化。

数据分析

为了定量带的密度,我们使用了MultiGauge(Fujifilm软件)。进行至少三次,优选4-6次独立实验用于统计分析。为了统计分析,我们使用了GraphPad Prism(GraphPad软件)。通常情况下,我们提供了一个代表性的印迹,用于在论文的主体统计分析和补充部分提供所有额外的印迹用于统计分析。由于条带密度被归一化为控制条带(例如总共SHP-1印迹),所有的数据应该是可用的并且包括在内,除非存在特定凝胶或印迹的技术问题。控制总蛋白质条带的标准化被认为是必要的,尤其是如果统计分析中包括单独的印迹。我们也发现凝胶(第一个和最后一个)上的边缘泳道可能是有问题的,所以我们尽量避免使用这两条泳道作为关键样本。参见Choi et al。(2017)对这些实验公布的数据的一个例子。

笔记

  1. 所有的溶液和培养基都应该用膜真空泵(37 L / min)脱气1小时,然后储存在冰上。

  2. 每种抗体的曝光时间会有所不同

食谱

  1. 氧化裂解缓冲液
    50 mM Tris(pH 7.5)
    100 mM NaCl
    0.1%SDS
    0.5%脱氧胆酸钠
    0.5%Nonidet P-40
    0.5%Triton X-100
    50 mM NaF
    1毫米PMSF
    0.4 mM碘乙酰PEG-生物素
    100μMDTPA
    200 U / ml过氧化氢酶
    每50毫升1片蛋白酶抑制剂
  2. 氧化清洗缓冲液
    50 mM Tris(pH 7.5)
    100 mM NaCl
    0.5%Nonidet P-40
    0.5%Triton X-100
    50 mM NaF
  3. TBST缓冲区
    20 mM Tris(pH 7.5)
    135 mM NaCl
    0.05%吐温20
  4. 1 mM pervanadate
    1mM Na 3 VO 4 4 5mM H 2 O 2:2 注意:使用前在室温下保温5分钟。
  5. 裂解缓冲液
    1%Nonidet P-40
    10 mM Tris(pH 7.5)
    150 mM NaCl
    2 mM EGTA
    50毫克β-甘油磷酸酯
    2 mM Na 3 VO 4 4 10 mM NaF
    10 mM碘乙酰胺
    10毫米NEM
    每50毫升1片蛋白酶抑制剂

致谢

国立儿童健康与人类发展研究所(EIA)的Eunice Kennedy Shriver室内研究计划(1ZIAHD001803至P.E.L.)支持这项工作。该方案已经改编自Choi em等人(2017)Nature Immunology 18,433-441。作者与本出版物没有利益冲突或利益冲突。

参考

  1. Choi,S.,Warzecha,C.,Zvezdova,E.,Lee,J.,Argenty,J.,Lesourne,R.,Aravind,L.和Love,P.E。(2017)。 THEMIS增强TCR信号传导,并通过选择性抑制磷酸酶SHP-1实现阳性选择。 Nat Immunol 18(4):433-441。
  2. Huyer,G.,Liu,S.,Kelly,J.,Moffat,J.,Payette,P.,Kennedy,B.,Tsaprailis,G.,Gresser,M.J。和Ramachandran,C。(1997)。 钒酸盐和过钒酸盐抑制蛋白酪氨酸磷酸酶的机制 J Biol Chem 272:843-51。
  3. Karisch,R.和Neel,B. G.(2013)。 监测经典蛋白酪氨酸磷酸酶氧化的方法 FEBS J 280(2):459-475。
  4. Persson,C.,Sjoblom,T.,Groen,A.,Kappert,K.,Engstrom,U.,Hellman,U.,Heldin,C.H.,den Hertog,J.and Ostman,A。(2004)。 氧化蛋白抗体所揭示的受体样PTP-α的第二磷酸酶结构域的优先氧化酪氨酸磷酸酶


    美国国家科学院院刊 101(7):1886-1891。

  5. Rudyk,O.和Eaton,P。(2014)。 在生物系统中监测蛋白质巯基氧化还原状态的生化方法 Redox Biol 2:803-813。
  6. Weibrecht,I.,Bohmer,S.A。,Dagnell,M.,Kappert,K.,Ostman,A。和Bohmer,F.D。(2007)。 蛋白酪氨酸磷酸酶SHP-1和SHP-2催化半胱氨酸的氧化敏感性 Free Radic Biol Med 43(1):100-110。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Choi, S. and Love, P. E. (2018). Detection of Intracellular Reduced (Catalytically Active) SHP-1 and Analyses of Catalytically Inactive SHP-1 after Oxidation by Pervanadate or H2O2. Bio-protocol 8(1): e2684. DOI: 10.21769/BioProtoc.2684.
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