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VAMP8-3xHA Uptake Assay in HeLa Cells

HeLa 细胞中的VAMP8-3xHA摄取试验

Steve Jean Steve Jean
Amy A. Kiger Amy A. Kiger

发布: 2016年02月20日第6卷第4期 DOI: 10.21769/BioProtoc.1739 浏览次数: 8640

评审: Ralph BottcherVaibhav B ShahAnonymous reviewer(s)

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Abstract

Transmembrane proteins are rarely exclusively localized to a specific vesicle or an organelle. Most transmembrane proteins undergo complicated trafficking routes. Thus, transmembrane proteins are under constant flux, and at steady state, found on a variety of vesicles or organelles. This characteristic makes the study of their trafficking routes complex, since at any given moment, different molecules are often being trafficked in opposing directions. Pulse-chase experiments can temporally track a specific pool of a transmembrane protein of interest, allowing for the kinetic description of its trafficking route. This type of technique has been used extensively to follow a large array of plasma membrane localized proteins (Diril et al., 2006; Jean et al., 2010). Here, we describe a method that allows the study of VAMP8 trafficking from the plasma membrane to endolysosomal compartments. This method was used to describe a role for MTMR13 and RAB21 in the regulation of VAMP8 trafficking to endolysosomes (Jean et al., 2015).

Keywords: Membrane trafficking (膜转运) search Endosomal sorting (胞内分选) search Autophagy (自噬) search

Materials and Reagents

  1. Costar® 24 Well Clear TC-Treated Well Plates (Corning, catalog number: 3526 ) 

  2. #1.5 round glass coverslip (Ted Pella, catalog number: 260368 ) 

  3. 100 mm Tissue Culture Dish (Corning, catalog number: 430167 ) 

  4. 0.22 µm filtering unit (Genesee Scientific Corporation, catalog number: 25-227 )
  5. HeLa cells (ATCC, catalog number: CCL-2 ) 

  6. JetPRIME (Polyplus-transfection, catalog number: 114-07 ) 

  7. pCDNA3-VAMP8-3xHA expression plasmid (self-made) (Jean et al., 2015)
  8. Dulbecco’s Modified Eagle Medium with High glucose with 4 mM L-Glutamine and sodium pyruvate (GE Healthcare, HycloneTM, catalog number: SH30243.FS ) (see Recipes for complete DMEM) 

  9. Fetal Bovine Serum (Sigma-Aldrich, catalog number: F2442-500 ml ) 

  10. Penicillin-Streptomycin solution (Life Technologies, catalog number: 15140-122 ) 

    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122”.
  11. Trypsin-EDTA (0.25%) (Life Technologies, catalog number: 25200-056 ) 

    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25200-056”.
  12. 0.4% Trypan-Blue (Life Technologies, catalog number: 15250-061 ) 

    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15250-061”.
  13. Rabbit anti-HA antibody (Abcam, catalog number: ab9110 ) 

  14. Earl’s Balanced Salt Solution (with sodium bicarbonate, without phenol red, liquid, sterile- filtered) (Sigma-Aldrich, catalog number: E3024-500 ml ) 

  15. Bovine Serum Albumin (Fraction V, Heat shock treated) (Thermo Fisher Scientific, catalog number: BP1600-100 ) 

  16. Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S671-3 )

  17. Potassium Chloride (KCl) (VWR International, catalog number: BDH9258-500 g ) 

  18. Sodium Phosphate Dibasic Anhydrous (Na2HPO4) (Thermo Fisher Scientific, catalog number: S374500 )

  19. Potassium Phosphate Monobasic (KH2PO4) (Thermo Fisher Scientific, catalog number: P285-S500 ) 

  20. Paraformaldehyde (Thermo Fisher Scientific, catalog number: AC41678-5000 ) 

  21. Goat Serum (Life Technologies, catalog number: 16210-064 ) 

    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 16210-064”.
  22. Triton X-100 (Sigma-Aldrich, catalog number: X100-500 ml ) 

  23. Mouse anti-human Lamp1 (Developmental Studies Hybridoma Bank, catalog number: H4A3 ) 

  24. Mouse anti-EEA1 (BD Biosciences, catalog number: 610457 ) 

  25. Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (Life Technologies, catalog number: A-11029 ) 

    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11029”.
  26. Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 546 conjugate (Life Technologies, catalog number: A11035 ) 

    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: A11035”.
  27. [4’, 6-Diamidino-2-Phenylindole, Dihydrochloride] (DAPI) (Life Technologies, catalog number: D1306 ) 

    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306”.
  28. FluorSave reagent (Merck Millipore Corporation, Calbiochem®, catalog number: 345789 ) 

  29. Complete DMEM (see Recipes) 

  30. 1x Phosphate Buffer Saline (PBS) (see Recipes) 

  31. 4% Paraformaldehyde solution (see Recipes) 

  32. Blocking buffer (see Recipes) 

  33. Antibody incubation buffer (see Recipes) 


Equipment

  1. 37 °C, 5% CO2 cell culture incubator [similar to Thermo Scientific Heracell VIOS 160i CO2 incubator (Thermo Fisher Scientific, catalog number: 51030285 )]

  2. Tissue culture biosafety cabinet [similar to Thermo Scientific 1300 Series Class II (Thermo Fisher Scientific, catalog number: 1323)] 

  3. Hemacytometer (VWR International, catalog number: 100498-470 ) 

  4. Pipettes 10 μl, 100 μl and 1,000 μl (Eppendorf, catalog numbers: 3120000020 , 3120000046 and 31200000623 )
  5. Vacuum Flask (self-made)
    Note: Required to remove media from the 24 well plates during the immunofluorescence protocol.
  6. Microcentrifuge (Eppendorf, catalog number: 5415D ) 


Procedure

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文章信息

版权信息

© 2016 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. Jean, S. and Kiger, A. A. (2016). VAMP8-3xHA Uptake Assay in HeLa Cells. Bio-protocol 6(4): e1739. DOI: 10.21769/BioProtoc.1739.
  2. Jean, S., Cox, S., Nassari, S. and Kiger, A. A. (2015). Starvation-induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome-lysosome fusion. EMBO Rep 16(3): 297-311.

    3. ris ris Download Citation in RIS Format

分类

细胞生物学 > 基于细胞的分析方法 > 转运

细胞生物学 > 细胞成像 > 共聚焦显微镜

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