Histone Deubiquitination Assay in Nicotiana benthamiana
本氏烟草中的组蛋白去泛素化分析   

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Nature Plants
Aug 2016

 

Abstract

Histone modifications are a group of post-translational modifications on histones which can alter chromatin structure and affect gene expression. Histone ubiquitination is a histone modification found in particular on histone H2A and H2B. Histone ubiquitination can be reversed by ubiquitin-specific proteases (UBP). Here, we describe an in vivo assay for histone deubiquitination activity. After infiltrating UBP12 into Nicotiana benthamiana leaves, H2Aub was visualized by immunocytochemistry. Nicotiana benthamiana leaves, which show high agro infiltration efficiency, were used for transient UBP12 expression for a labor- and time-saving protocol. Reduced H2Aub levels indicated histone deubiquitination activity of UBP12. The clear visualization of nuclei of N. benthamiana leaves makes this method able to easily measure the level of histone modification in vivo by using specific antibodies, providing robust clues of protein function. Thus, this protocol is a powerful complementation to in vitro assays of histone deubiquitination activity.

Keywords: Histone deubiquitination (组蛋白去泛素化), Immunocytochemistry (免疫细胞组化), H2Aub (H2Aub), in vivo (体内试验)

Background

Histone modifications play important roles in regulating chromatin structure and gene expression. Best studied histone modifications include methylation, acetylation, phosphorylation, ubiquitination and sumoylation. However, enzymes introducing or removing specific histone modifications are not always known. Powerful in vitro assays can establish the catalytic potential of histone modifying enzymes but in vivo methods are desirable to confirm that in vitro specificity reflects in vivo activity. Here, we describe a flexible protocol to test activity of histone modifying enzymes in the plant N. benthamiana. Although we used the protocol to test activity of ubiquitin specific protease (UBP) on ubiquitylated H2A, it can also easily be adopted to other histone modifications for which specific antibodies are available.

Materials and Reagents

  1. 50 ml and 15 ml screw cap tubes (SARSTEDT, catalog numbers: 62.547.254 and 62.554.002 )
  2. 2 ml and 1.5 ml microtubes (SARSTEDT, catalog numbers: 72.695.500 and 72.690.001 )
  3. 5 ml syringe (BD plastipak, BD, catalog number: 302187 )
  4. Razor blade (Feather Safety Razor, catalog number: 02.015.00.024 )
  5. Petri dish, 9.2 cm diameter (SARSTEDT, catalog number: 82.1473.001 )
  6. CellTrics® (pore size 30 μm) (Sysmex, catalog number: 04-0042-2316 )
  7. Absorbent paper (Kimberly-Clark, catalog number: 3020 )
  8. Microscopic slides and cover slips (VWR, catalog number: 631-1551 and 631-9430 )
  9. Nicotiana benthamiana wild-type plants (seeds kindly provided by Dr. Savenkov, Uppsala), 4-6 weeks old (cultivated at 24 °C,16 h light, 8 h dark, 60% humidity); mature leaves (flat surface and edge) are used
  10. Agrobacterium tumefaciens strain GV3101 harboring pUBC-UBP12-CFP plasmid (Derkacheva et al., 2016)
  11. Agrobacterium tumefaciens strain GV3101 harboring pUBC-H3.3-CFP plasmid (Derkacheva et al., 2016)
  12. Agrobacterium tumefaciens strain GV3101 harboring viral RNA silencing suppressor p19 (kindly provided by E. Savenkov, Uppsala)
  13. LB broth high salt (Duchefa Biochemie, catalog number: L1704.2500 )
  14. Formaldehyde (Sigma-Aldrich, catalog number: F8775 )
  15. Antibody anti-H2Aub (Cell Signaling Technology, catalog number: 8240 )
  16. Antibody anti-H4 (Merck, EMD Millipore, catalog number: 05-858 )
  17. Antibody Alexa Fluor 555 conjugated goat anti-rabbit Kit (Thermo Fisher Scientific, Invitrogen, catalog number: A31629 )
  18. VECTASHIELD® mounting medium with 1 μg/ml DAPI (Vector Laboratories, catalog number: H-1200 )
  19. Bovine serum albumin (BSA) (Carl Roth, catalog number: 3737.2 )
  20. MES (Duchefa Biochemie, catalog number: M1503 )
  21. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M9272 )
  22. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
  23. Tris base (VWR, catalog number: 28811.364 )
  24. EDTA disodium salt (Na2-EDTA) (Sigma-Aldrich, catalog number: ED2SS )
  25. Sodium chloride (NaCl) (Merck, catalog number: 567441 )
  26. 37% HCl (Merck, catalog number: 1.00317.2500 )
  27. Spermine (Sigma-Aldrich, catalog number: 85590 )
  28. Potassium chloride (KCl) (Merck, catalog number: 529552 )
  29. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  30. Sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) (Merck, EMD Millipore, catalog number: 106342 )
  31. Sodium hydrogen phosphate (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
  32. Infiltration medium (see Recipes)
  33. Tris buffer (see Recipes)
  34. LB01 lysis buffer (see Recipes)
  35. PBS buffer (see Recipes)

Equipment

  1. Centrifuges (Eppendorf, model: 5804 R and Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM PicoTM 17 )
  2. Surgical scissors (DIMEDA Instrumente, catalog number: 08.340.11 )
  3. Laminar hood
  4. Pipettes (Gilson, model: PIPETMAN® Classic, P2 , P20 , P200 , P1000 )
  5. Humid incubator
  6. Confocal microscope (ZEISS, model: LSM 780 )

Procedure

  1. Culture A. tumefaciens carrying pUBC-UBP12-CFP, pUBC-H3.3-CFP (as a control) or p19 in 3 ml of liquid LB in 15 ml screw cap tubes separately for 20 h at 28 °C.
  2. Centrifuge the culture in 2 ml microtubes at 1,500 x g for 5 min at room temperature, discard the supernatant and then suspend the A. tumefaciens pellet in infiltration medium by adjusting OD600 to 1.0.
  3. Combine the same amount (usually 1 ml) of pUBC-UBP12-CFP or pUBC-H3.3-CFP with (1 ml) p19 culture in a 2 ml microtube.
    Note: The p19 construct can increase expression of transgenes in transfected N. benthamiana cells. Without p19 expression, transfected N. benthamiana cells often silence transgenes resulting in low and variable expression. Presence of p19 partially blocks RNA silencing and thus establishes higher and more uniform transgene expression making the presented assay more sensitive and more reproducible.
  4. Place the microtube horizontally for 2 h at room temperature.
  5. Use a needle to punch some holes in the abaxial (lower) side of 3 to 4 N. benthamiana leaves and infiltrate the A. tumefaciens culture with a 5 ml syringe.
  6. Between 48 h and 72 h after infiltration, the area of the infiltrated tobacco leaves near the holes is cut into pieces of about 1 x 1 cm using scissors and fixed in 10 ml of Tris buffer containing 4% formaldehyde in 50 ml screw cap tubes on ice for 20 min (operate in a laminar hood to avoid exposure to formaldehyde).
  7. Pour off the Tris buffer and wash the sample twice with 20 ml of ice-cold Tris buffer each for 10 min.
  8. Chop the leaves with a razor blade in 400 μl of LB01 buffer in a Petri dish on ice to release the nuclei.
  9. Filter the suspension through CellTrics® into a new 1.5 ml microtube.
  10. Pipet 5 to 10 μl of the suspension onto a microscopic slide and air-dry the suspension (about 5 to 10 min) (Note 1).
  11. Pipette 100 μl of PBS buffer containing 4% formaldehyde onto the slides to postfix the nuclei for 30 min at room temperature (operate in a laminar hood to avoid exposure to formaldehyde), then lean the slides and absorb the PBS buffer by absorbent paper (Note 2).
  12. Pipette 100 μl of PBS buffer onto the slides to cover the nuclei for rinsing 5 min, then lean the slides and absorb the PBS buffer by absorbent paper, repeat once.
  13. Permeabilize the cells with 100 μl of 0.2% Triton X-100 (diluted in PBS buffer) for 5 min, then lean the slides and absorb the PBS buffer by absorbent paper.
  14. Pipette 100 μl of PBS buffer onto the slides to rinse the samples, then lean the slides and absorb the PBS buffer by absorbent paper, repeat 3-4 times.
  15. Apply sufficient volume (20-100 μl) of Image-iTTM FX signal enhancer to cover nuclei. Incubate for 30 min at room temperature in a humid incubator (Notes 3 and 4).
  16. Pipette 100 μl of PBS buffer onto the slides, then lean the slides and absorb the PBS buffer by absorbent paper.
  17. Antibody detection: Mix 1 μl of anti-H2Aub or anti-H4 (as a negative control) with 100 μl of 1% BSA in PBS buffer, pipet the solution on the slides, and incubate in a humid incubator at 37 °C for 2 h (Note 4).
  18. Pipette 100 μl of PBS buffer onto the slides to rinse the nuclei at room temperature three times, each for 10 min. Lean the slides and absorb the PBS buffer with absorbent paper each time.
  19. Mix 5 μl of Alexa Fluor 555 conjugated goat anti-rabbit antibody with 1 ml of 1% BSA in PBS buffer, pipet the solution onto the slides and incubate at 37 °C for 30 min.
  20. Pipette 100 μl of PBS buffer onto the slides at room temperature three times, each for 10 min. Lean the slides and absorb the PBS buffer with absorbent paper each time.
  21. Pipette VECTASHIELD® mounting medium with 1 μg/ml DAPI onto microscopic slides and cover with cover slips.
  22. Observe nuclei (DAPI), CFP signal and antibody signal under a confocal microscope such as a ZEISS LSM 780 using appropriate excitation wavelength. DAPI is excited by the 405 nm laser line; CFP is detected by the 485 nm laser line, while antibody is excited by the 555 nm laser line. DAPI shows the position of nuclei and CFP labels nuclei from transfected cells. Check several fields (about 50 nuclei) and compare the difference when infiltrating N. benthamiana leaves with A. tumefaciens carrying pUBC-UBP12-CFP or pUBC-H3.3-CFP. Figure 1 shows examples of microscopic detection of transfected nuclei and immunocytochemistry for the analyzed histone posttranslational modification (PTM; here, H2Aub). We found that H2Aub signals significantly decreased in cells expressing UBP12 compared to control cells expressing H3.3 (Figure 1A). In contrast, H4 signals did not differ significantly when UBP12 or H3.3 were expressed in N. benthamiana leaves (Figure 1B), implying deubiquitination activity of UBP12.


    Figure 1. Examples of assay results–UBP12 has deubiquitinase activity. A. Infiltration of pUBC-UBP12-CFP but not of pUBC-H3.3-CFP reduced H2A ubiquitylation in N. benthamiana leaves. B. Infiltration of either pUBC-H3.3-CFP or pUBC-UBP12-CFP did not affect H4 signals in N. benthamiana leaves. Scale bars = 30 µm.

Data analysis

Check several fields containing together at least 50 nuclei for statistical analysis. Choose fields with similar background signal because some regions on the slide may have high background from inadequate rinsing. Figure 2 shows a strategy for quantitative analysis of assay results.
Look for nuclei by DAPI signal (Figures 1A and 1B, left panel) and use the CFP signal to select nuclei that were successfully transfected with pUBC-H3.3-CFP (control construct) or pUBC-UBP12-CFP (test construct) (Figures 1A and 1B, center panel). Compare transfection rates (Figure 2A); ideally, transfection rates should be similar for control and test constructs. Count the number of nuclei with fluorescent signals for H2Aub or H4 (Figures 1A and 1B, right panel). Detection efficiency of immunocytochemistry can be expressed as number of H4-positive nuclei as percentage of all transfected nuclei (Figure 2B). Similar values indicate that the detection process (fixing, staining, washing etc.) was comparable for control and test constructs. The effects of control and test constructs on target PTM can be seen in a comparison of the percentages of PTM-positive nuclei after transfection (Figure 2C). Smaller values for the test than for the control construct indicate that the test construct specifically reduced the target PTM. An alternative display is to compare the relative frequencies of PTM-positive nuclei (Figure 2D). For this, the frequency of PTM-positive nuclei after transfection with the control construct is set as 1 and the frequency of PTM-positive nuclei after transfection with the test construct is expressed relative to it. A well-working test construct should affect the target PTM and thus show a relative frequency of PTM-positive nuclei < 1. This display directly shows the effect size of the transfected test construct and is recommended when transfection or detection rates differ; results should be compared across several experiments or when multiple test constructs are included.


Figure 2. Strategy for quantitative analysis of assay results for a histone posttranslational modification (PTM; here: H2Aub). Control and test constructs (here: pUBC-H3.3-CFP and pUBC-UBP12-CFP) are represented by light grey and dark grey bars, respectively. A. Transfection rate, i.e., number of CFP-positive nuclei as percentage of all detected nuclei. Similar bar heights indicate similar transfection rates for control and test constructs. B. Detection efficiency of immunocytochemistry, i.e., number of H4-positive nuclei as percentage of all transfected nuclei. Similar bar heights indicate that the detection process (fixing, staining, washing etc.) was similar for control and test constructs. C. Effect of control and test construct on target PTM, i.e., number of PTM-positive nuclei as percentage of all transfected nuclei. Smaller values for the test than for the control construct indicate that the test construct specifically reduced the target PTM. D. Alternative display of the effect of control and test constructs on target PTM using relative frequencies. The frequency of PTM-positive nuclei transfected with the pUBC-H3.3-CFP control construct was set to 1 and the frequency of PTM-positive nuclei transfected with the pUBC-UBP12-CFP test construct expressed relative to this. A well-working test construct (here: pUBC-UBP12-CFP) should affect the target PTM and thus result in a relative intensity < 1.

Notes

  1. After air dry, white contents can be seen on the slides. The following steps are all performed on the slides. Make sure the liquid covers the white contents.
  2. Slides can be put in an empty pipette tip box to keep them moist.
  3. Image-iTTM FX signal enhancer is in the same kit with Alexa Fluor 555 conjugated goat anti-rabbit antibody. It is not intended for use with live cells.
  4. An empty pipette tip box with some water is used as a humid incubator.
  5. Check several times to make sure the antibody solution continuously covers nuclei, if not, add more antibody solution.

Recipes

  1. Infiltration medium (freshly prepared)
    10 mM MES (pH 5.7)
    10 mM MgCl2
    150 μM acetosyringone
  2. Tris buffer
    10 mM Tris-HCl (pH 7.5) (see Recipe 3)
    10 mM Na2-EDTA
    100 mM NaCl
  3. 1 M Tris-HCl (pH 7.5)
    121 g Tris base in 1 L H2O
    Adjust pH with 37% HCl
  4. LB01 lysis buffer
    15 mM Tris-HCl (pH 7.5)
    2 mM Na2-EDTA
    0.5 mM spermine
    80 mM KCl
    20 mM NaCl
    0.1% Triton X-100
  5. PBS buffer
    10 mM sodium phosphate (pH 7.0) (see Recipe 6)
    143 mM NaCl
  6. 1 M sodium phosphate (pH 7.0)
    5.77 ml 1 M NaH2PO4
    4.23 ml 1 M Na2HPO4

Acknowledgments

Seeds of N. benthamiana and the construct harboring viral RNA silencing suppressor p19 were kindly provided by E. Savenkov (SLU, Uppsala). This protocol was developed from the following published paper: Derkacheva et al. (2016). This work was supported by a grant from the Knut-and-Alice-Wallenberg Foundation. The authors declare no conflicts of interest or competing interests.

References

  1. Derkacheva, M., Liu, S., Figueiredo, D. D., Gentry, M., Mozgova, I., Nanni, P., Tang, M., Mannervik, M., Kohler, C. and Hennig, L. (2016). H2A deubiquitinases UBP12/13 are part of the Arabidopsis Polycomb group protein system. Nat Plants 2: 16126.

简介

组蛋白修饰是一组组蛋白翻译后修饰,可以改变染色质结构并影响基因表达。组蛋白泛素化是组蛋白H2A和H2B特异性发现的组蛋白修饰。泛素特异性蛋白酶(UBP)可以逆转组蛋白泛素化。在这里,我们描述了组蛋白去泛素化活性的体内试验。在将UBP12渗入烟草叶片中后,通过免疫细胞化学观察H2Aub。表现出高的农业渗透效率的本氏烟草叶用于瞬时UBP12表达,用于节省劳力和时间的方案。 H2Aub水平降低表明UBP12的组蛋白去泛素化活性。 N的核的清晰可视化。本生叶使得该方法能够通过使用特异性抗体容易地测量体内组蛋白修饰的水平,从而提供强大的蛋白质功能线索。因此,该协议是组蛋白去泛素化活性的体外试验的有力补充。

【背景】组蛋白修饰在调节染色质结构和基因表达中发挥重要作用。 研究最深入的组蛋白修饰包括甲基化,乙酰化,磷酸化,泛素化和sumoylation。 然而,引入或去除特定组蛋白修饰的酶并不总是已知的。 强大的体外试验可以确定组蛋白修饰酶的催化潜能,但是体内试验方法对于确认体外试验的特异性反映抗体的特异性是必要的。 体内活动。 在这里,我们描述了一个灵活的协议来测试植物组织中组蛋白修饰酶的活性。本塞姆氏。 尽管我们使用该方案来测试遍在蛋白特异性蛋白酶(UBP)对泛素化H2A的活性,但它也可以容易地用于其他特异性抗体可用的组蛋白修饰。

关键字:组蛋白去泛素化, 免疫细胞组化, H2Aub, 体内试验

材料和试剂

  1. 50毫升和15毫升螺旋盖管(SARSTEDT,产品目录号:62.547.254和62.554.002)
  2. 2毫升和1.5毫升微管(SARSTEDT,目录号:72.695.500和72.690.001)
  3. 5毫升注射器(BD plastipak,BD,目录号:302187)
  4. 剃须刀片(羽毛安全剃须刀,目录号:02.015.00.024)
  5. 培养皿,9.2厘米直径(SARSTEDT,目录号:82.1473.001)
  6. CellTrics (孔径30μm)(Sysmex,目录号:04-0042-2316)
  7. 吸水纸(Kimberly-Clark,目录号:3020)
  8. 显微镜载玻片和盖玻片(VWR,目录号:631-1551和631-9430)
  9. (4周龄,16小时光照,8小时黑暗,60%湿度下培养)的野生型植物(由Savenkov博士,乌普萨拉友好提供的种子)烟草本生烟草野生型植物成熟的叶子(平坦的表面和边缘)被使用
  10. 含有pUBC-UBP12-CFP质粒的根瘤土壤杆菌菌株GV3101(Derkacheva等人,2016)
  11. 含有pUBC-H3.3-CFP质粒的根癌土壤杆菌菌株GV3101(Derkacheva等人,2016)
  12. 含有病毒RNA沉默抑制子p19的根癌土壤杆菌菌株GV3101(由E.Savankov,Uppsala友情提供)
  13. LB肉汤高盐(Duchefa Biochemie,目录号:L1704.2500)
  14. 甲醛(Sigma-Aldrich,目录号:F8775)
  15. 抗体抗H2Aub(Cell Signaling Technology,目录号:8240)
  16. 抗体抗-H4(Merck,EMD Millipore,目录号:05-858)
  17. 抗体Alexa Fluor 555结合山羊抗兔试剂盒(Thermo Fisher Scientific,Invitrogen,目录号:A31629)
  18. 带有1μg/ ml DAPI(Vector Laboratories,产品目录号:H-1200)的VECTASHIELD®安装介质。
  19. 牛血清白蛋白(BSA)(Carl Roth,目录号:3737.2)
  20. MES(Duchefa Biochemie,目录号:M1503)
  21. 氯化镁六水合物(MgCl 2•6H 2 O)(Sigma-Aldrich,目录号:M9272)
  22. 乙酰丁香酮(Sigma-Aldrich,目录号:D134406)
  23. Tris碱(VWR,目录号:28811.364)
  24. EDTA二钠盐(Na 2 -EDTA)(Sigma-Aldrich,目录号:ED2SS)
  25. 氯化钠(NaCl)(Merck,目录号:567441)
  26. 37%HCl(Merck,目录号:1.00317.2500)
  27. 精胺(Sigma-Aldrich,目录号:85590)
  28. 氯化钾(KCl)(Merck,目录号:529552)
  29. Triton X-100(Sigma-Aldrich,目录号:X100)
  30. 磷酸二氢钠二水合物(NaH 2 PO 4•2H 2 O)(Merck,EMD Millipore,目录号:106342)
  31. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S3264)
  32. 渗透介质(见食谱)
  33. Tris缓冲液(见食谱)
  34. LB01裂解缓冲液(见食谱)
  35. PBS缓冲液(见食谱)

设备

  1. 离心机(Eppendorf,型号:5804R和Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Pico TM 17)
  2. 手术剪(DIMEDA Instrumente,目录号:08.340.11)
  3. 层流罩
  4. 移液器(Gilson,型号:PIPETMAN®Classic Classic,P2,P20,P200,P1000)
  5. 湿培养箱
  6. 共聚焦显微镜(ZEISS,型号:LSM 780)

程序

  1. 文化 A。分别在15ml螺旋盖管中的3ml液体LB中在28℃下将pUBC-UBP12-CFP,pUBC-H3.3-CFP(作为对照)或p19持续20小时, >
  2. 在室温下将培养物在2ml微管中离心5分钟5分钟,丢弃上清液,然后通过调节OD 将悬浮在渗透培养基中的根瘤农杆菌沉淀物> 600 至1.0。
  3. 将相同量(通常1ml)的pUBC-UBP12-CFP或pUBC-H3.3-CFP与(1ml)p19培养物在2ml微管中合并。
    注意:p19构建体可以增加转染的本氏烟草细胞中转基因的表达。没有p19表达,转染的本氏烟草细胞经常沉默转基因,导致低表达和可变表达。 p19的存在部分阻断RNA沉默,从而建立更高和更均匀的转基因表达,使得所提供的测定更灵敏并且更可重复。

  4. 在室温下将微管水平放置2小时。
  5. 用针在3至4em的背面(下)侧打孔。使用5毫升注射器将本生叶培养物浸入土壤杆菌培养物中。
  6. 在渗透后48小时和72小时之间,使用剪刀将渗入的孔附近的烟草叶片的面积切成约1×1cm的片,并在10ml含有4%甲醛的Tris缓冲液中在50ml螺帽管中固定冰20分钟(在层流罩中操作以避免暴露于甲醛)。
  7. 倒出Tris缓冲液,用20ml冰冷的Tris缓冲液洗涤样品两次,每次10分钟。

  8. 在冰上的培养皿中用400μlLB01缓冲液将叶片用刀片切下,以释放核。
  9. 通过CellTrics ®将悬浮液过滤到新的1.5 ml微管中。
  10. 吸取5到10微升悬浮液到显微镜载玻片上并风干悬浮液(约5到10分钟)(注1)。
  11. 吸取100μl含有4%甲醛的PBS缓冲液至载玻片上以在室温下在核后30分钟(在层流罩中操作以避免暴露于甲醛),然后倾斜载玻片并用吸收纸吸收PBS缓冲液(注意2)。
  12. 吸取100μlPBS缓冲液到载玻片上以覆盖细胞核漂洗5分钟,然后倾斜载玻片并用吸水纸吸收PBS缓冲液,重复一次。
  13. 用100μl0.2%Triton X-100(在PBS缓冲液中稀释)使细胞透化5分钟,然后倾斜玻片并用吸水纸吸收PBS缓冲液。
  14. 吸取100μlPBS缓冲液到载玻片上冲洗样品,然后倾斜玻片并用吸水纸吸收PBS缓冲液,重复3-4次。
  15. 使用足够的体积(20-100μl)Image-iT TM TM FX信号增强剂覆盖原子核。在潮湿的培养箱中孵育30分钟(注3和4)。
  16. 吸取100μlPBS缓冲液到载玻片上,然后倾斜载玻片并用吸水纸吸收PBS缓冲液。
  17. 抗体检测:将1μl抗H2Aub或抗H4(作为阴性对照)与100μl1%BSA的PBS缓冲液混合,将溶液吸到载玻片上,并在37℃的潮湿培养箱中孵育2 h(注4)。
  18. 吸取100μlPBS缓冲液到载玻片上,在室温下冲洗细胞核三次,每次10分钟。倾斜玻片,每次吸水纸吸收PBS缓冲液。
  19. 将5μlAlexa Fluor 555结合的山羊抗兔抗体与1 ml 1%BSA的PBS缓冲液混合,将溶液吸到载玻片上并在37℃孵育30分钟。
  20. 在室温下将100μlPBS缓冲液移至载玻片上三次,每次10分钟。倾斜玻片,每次吸水纸吸收PBS缓冲液。
  21. 用含有1μg/ ml DAPI的VECTASHIELD®安装介质移液到显微镜载玻片上并盖上盖玻片。
  22. 使用适当的激发波长,在共焦显微镜如ZEISS LSM 780下观察细胞核(DAPI),CFP信号和抗体信号。 DAPI由405 nm激光线激发; 485nm激光线检测CFP,而555nm激光线激发抗体。 DAPI显示来自转染细胞的细胞核和CFP标记核的位置。检查几个领域(大约50个核),并比较渗透 benthamiana 叶与 A时的差异。携带pUBC-UBP12-CFP或pUBC-H3.3-CFP的根癌农杆菌。图1显示转染细胞核的显微镜检测和分析的组蛋白翻译后修饰(PTM;此处为H2Aub)的免疫细胞化学实例。我们发现与表达H3.3的对照细胞相比,在表达UBP12的细胞中H2Aub信号显着降低(图1A)。相反,当UBP12或H3.3在N中表达时,H4信号没有显着差异。本氏烟叶(图1B),暗示UBP12的去泛素化活性。


    图1.测定结果实例 - UBP12具有去泛素化酶活性。 :一种。 pUBC-UBP12-CFP而不是pUBC-H3.3-CFP的渗透降低了N中的H2A泛素化。本生烟叶叶。 B.pUBC-H3.3-CFP或pUBC-UBP12-CFP的渗入不影响本氏烟草叶中的H4信号。比例尺= 30微米。

数据分析

检查包含至少50个核的几个域以进行统计分析。选择具有相似背景信号的区域,因为载玻片上的某些区域可能因冲洗不足而产生高背景。图2显示了测定结果定量分析的策略。
通过DAPI信号寻找细胞核(图1A和1B,左图)并使用CFP信号来选择用pUBC-H3.3-CFP(对照构建体)或pUBC-UBP12-CFP(测试构建体)成功转染的细胞核(图1A和1B,中心图)。比较转染率(图2A);理想情况下,对照和测试构建体的转染率应该相似。用H2Aub或H4的荧光信号计数核数(图1A和1B,右图)。免疫细胞化学的检测效率可以表示为H4阳性细胞核的数量作为所有转染细胞核的百分比(图2B)。类似的值表明对照和测试构建体的检测过程(固定,染色,洗涤等等)是可比较的。对照和测试构建体对靶PTM的作用可以在转染后PTM阳性细胞核百分比的比较中看到(图2C)。测试值小于对照构建体,表明测试构建体特异性地降低了目标PTM。另一种显示是比较PTM阳性细胞核的相对频率(图2D)。为此,用对照构建体转染后PTM阳性细胞核的频率设定为1,并且用测试构建体转染后PTM阳性细胞核的频率相对于其表达。运作良好的测试构建体应该影响目标PTM,并因此显示PTM-阳性核的相对频率1.此显示直接显示转染或检测率不同时推荐的转染测试构建体的效应大小,结果应该在多个实验中进行比较,或者包含多个测试结构。


图2.组蛋白翻译后修饰(PTM;此处为H2Aub)测定结果的定量分析策略。对照和测试构建体(这里:pUBC-H3.3-CFP和pUBC-UBP12-CFP)分别由浅灰色和深灰色条表示。 A.转染率,即,CFP阳性核的数量占所有检测到的核的百分比。类似的杆高表明对照和测试构建体的转染率相似。 B.免疫细胞化学检测效率,即,H4阳性细胞核的数量占所有转染细胞核的百分比。类似的杆高表明对照和测试构建体的检测过程(固定,染色,洗涤等等)是相似的。 C.对照和测试构建体对靶PTM的影响,即PTM阳性细胞核的数量占所有转染细胞核的百分比。测试值小于对照构建体,表明测试构建体特异性地降低了目标PTM。 D.使用相对频率替代显示对照和测试构建体对目标PTM的影响。将用pUBC-H3.3-CFP对照构建体转染的PTM阳性细胞核的频率设定为1,并且用pUBC-UBP12-CFP测试构建体转染的PTM阳性细胞核的频率相对于此被表达。运行良好的测试构建体(这里:pUBC-UBP12-CFP)应该影响靶PTM并因此导致相对强度< 1。

笔记

  1. 空气干燥后,幻灯片上可以看到白色的内容。以下步骤都在幻灯片上执行。确保液体覆盖白色内容。
  2. 幻灯片可以放在一个空的移液器吸头盒中,以保持湿润。
  3. Image-iT TM TM FX信号增强子与Alexa Fluor 555缀合的山羊抗兔抗体在相同的试剂盒中。它不适用于活细胞。
  4. 一个带有一些水的空吸管尖端盒被用作潮湿的培养箱。
  5. 检查几次,以确保抗体溶液连续覆盖细胞核,如果不是,则添加更多抗体溶液。

食谱

  1. 渗透介质(新鲜制备)
    10 mM MES(pH 5.7)
    10mM MgCl 2 2/2 150μM乙酰丁香酮
  2. Tris缓冲液
    10 mM Tris-HCl(pH 7.5)(见配方3)
    10mM Na 2 -EDTA
    100 mM NaCl
  3. 1M Tris-HCl(pH 7.5)
    在1升H 2 O中的121克Tris碱 用37%的HCl调节pH值
  4. LB01裂解缓冲液
    15 mM Tris-HCl(pH 7.5)
    2mM Na 2 -EDTA
    0.5毫米精胺
    80 mM KCl
    20 mM NaCl
    0.1%Triton X-100
  5. PBS缓冲液
    10mM磷酸钠(pH7.0)(见配方6)
    143 mM NaCl
  6. 1M磷酸钠(pH 7.0)
    5.77毫升1M NaH 2 PO 4水溶液 4.23ml 1M Na 2 HPO 4 4

致谢

N的种子。本氏烟草和含有病毒RNA沉默抑制因子p19的构建体由E.Savenkov(SLU,Uppsala)友情提供。该协议是从以下发表的论文开发的:Derkacheva et al。(2016)。这项工作得到了Knut-and-Alice-Wallenberg基金会的资助。作者声明不存在利益冲突或利益冲突。

参考

  1. Derkacheva,M.,Liu,S.,Figueiredo,DD,Gentry,M.,Mozgova,I.,Nanni,P.,Tang,M.,Mannervik,M.,Kohler,C。和Hennig,L。 )。 H2A去泛素化酶UBP12 / 13是拟南芥Polycomb组蛋白质系统的一部分。 Nat植物 2:16126。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, S. and Hennig, L. (2018). Histone Deubiquitination Assay in Nicotiana benthamiana. Bio-protocol 8(5): e2746. DOI: 10.21769/BioProtoc.2746.
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