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Secretion of Adipsin as an Assay to Measure Flux from the Endoplasmic Reticulum (ER)

脂肪酶分泌测定作为内质网（ER）流量测定法

AB Alexandria Brumfield
NC Natasha Chaudhary
TM Timothy E. McGraw

发布: 2017年04月05日第7卷第7期 DOI: 10.21769/BioProtoc.2204 浏览次数: 8945

评审: Ralph BottcherMirko Messamarzia Di DI DONATO

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Abstract

In this protocol we describe a quantitative biochemical assay to assess the efficiency of endoplasmic reticulum (ER) to Golgi protein transport in adipocytes (Bruno et al., 2016). The assay takes advantage of the fact that adipocytes secrete various bioactive proteins, known as adipokines. As a measure of ER to Golgi flux we determine the rate of bulk secretion of the adipokine adipsin post washout of Brefeldin A (BFA) treatment using immunoblotting. Because BFA treatment results in an accumulation of adipsin in the ER, the exit of adipsin from the ER upon BFA washout is synchronized across cells and experimental conditions. Thus, using this simple assay one can robustly determine if perturbations, such as knocking down a protein, have an effect on ER to Golgi protein transport.

Keywords: ER secretion (ER分泌) search Secretory pathway (分泌途径) search Adipocytes (脂肪细胞) search Adipsin (脂肪酶) search Brefeldin A (布雷非德菌素A) search

Background

Newly synthesized proteins destined to be secreted from the cell traffic through the secretory pathway to the plasma membrane (PM). The secretory route includes transport from the ER to the Golgi, across the Golgi stacks, and movement from the trans Golgi network (TGN) to the PM. Each of these transport steps provides nodes for regulation of secretion. While most cells are capable of secreting proteins, certain specialized cell types are professional secreters of specific proteins. Adipocytes, for example, secrete hormones, called adipokines that affect the energy metabolism of various organs. To better understand the molecular underpinnings of adipokine secretion, we have developed an assay to study the transport of adipsin, an adipokine, from the ER to the Golgi of cultured adipocytes. A traditional and commonly used method of studying ER to Golgi transport is to monitor the exit of the temperature sensitive vesicular stomatitis virus G protein ts045 fused to GFP (VSVG-GFP) from the ER (Presley et al., 1997). An advantage of using the adipsin secretion assay to study ER to Golgi transport in adipocytes is that the flux of an endogenous protein from the ER is monitored rather than an ectopically-expressed reporter protein.

Materials and Reagents

  1. 24-well cell culture plates (Corning, Falcon®, catalog number: 353226 )
  2. 10 cm dish
  3. 1.5 ml microcentrifuge tubes
  4. Pipette tips 
  5. 26 G ½ needles (BD, catalog number: 305111 )
  6. Cell scrapers (Corning, Falcon®, catalog number: 353085 )
  7. 3T3-L1 fibroblast cells (ATCC, Clone-173 )
  8. DMEM/10% FBS (see Recipes)
    1. Dulbecco’s modified Eagle medium (DMEM) powder high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 12100046 )
    2. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140095 )
    3. Penicillin-streptomycin (5,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15070063 )
    4. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S6297 )
  9. Brefeldin A (BFA) (5 μg/ml, prepared in M-199 media) (Cell Signaling, catalog number: 9972 )
  10. M-199 protein free media (Sigma-Aldrich, catalog number: M4530 )
  11. Acetone (cooled at -20 °C) (Sigma-Aldrich, catalog number: 320110 )
  12. Laemmli sample buffer (LSB) (2x, dilute in water for 1x) (Sigma-Aldrich, catalog number: S3401 )
  13. Trichloroacetic acid (TCA) (chilled on ice) (Sigma-Aldrich, catalog number: T0699 )
  14. 10x stock solution of phosphate-buffer saline (PBS) (see Recipes)
    1. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625 )
    2. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P4504 )
    3. Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Sigma-Aldrich, catalog number: S9390 )
    4. Monopotassium phosphate (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
  15. 1x stock solution of phosphate-buffer saline (PBS) (see Recipes)
  16. Polyacrylamide gels (10%) (home-made) (see Recipes)
    1. 30% Acrylamide/Bis solution 37.5:1 (Bio-Rad Laboratories, catalog number: 1610158 )
    2. Tris (Sigma-Aldrich, catalog number: T6066 )
    3. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 )
    4. Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678 )
    5. Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281 )
  17. Non-fat powdered milk (LabScientific, catalog number: M-0842 )
  18. Tween-20 (Sigma-Aldrich, catalog number: P1379 )
  19. Goat anti-adipsin antibody (Santa Cruz Biotechnology, catalog number: sc-12402 )
  20. HRP conjugated Donkey anti-Goat secondary antibody (Santa Cruz Biotechnology, catalog number: sc-2020 )
  21. 10x running buffer & transfer buffer for Western blotting (see Recipes)
    1. Glycine (Sigma-Aldrich, catalog number: G8898 )
    2. Tris
    3. Sodium dodecyl sulfate (SDS)
    4. Ethanol (EtOH) (Decon Labs, catalog number: V1016 )

Equipment

  1. Pipettes 
  2. Incubator at 37 °C/5% CO2
  3. Microcentrifuge at 4 °C (Thermo Fisher Scientific, model: HeraeusTM BiofugeTM StratosTM Centrifuge Series , catalog number: 75003236)
  4. Heat block at 95 °C
  5. Aspirator
  6. Western blotting equipment
    1. Mini Trans-Blot Cell and PowerPac Basic Power Supply (Bio-Rad Laboratories, catalog number: 1703989 )
    2. Nitrocellulose membrane (Nitrobind, 0.45 μm) (VWR, catalog number: 490007-490 )
    3. Blotting paper (VWR, catalog number: 28298-030 )

    Software

    1. ImageJ

    Procedure

    English
    中文翻译

    文章信息

    版权信息

    © 2017 The Authors; exclusive licensee Bio-protocol LLC.

    如何引用

    Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

    1. Brumfield, A., Chaudhary, N. and McGraw, T. E. (2017). Secretion of Adipsin as an Assay to Measure Flux from the Endoplasmic Reticulum (ER). Bio-protocol 7(7): e2204. DOI: 10.21769/BioProtoc.2204.
    2. Bruno, J., Brumfield, A., Chaudhary, N., Iaea, D. and McGraw T. E. (2016). SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes. J Cell Biol 214: 61-76.

      3. ris ris Download Citation in RIS Format

    分类

    生物化学 > 蛋白质 > 定量

    细胞生物学 > 基于细胞的分析方法 > 转运

    细胞生物学 > 细胞新陈代谢 > 其它化合物

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