|RIPA buffer||Life Technologies||89900|
|protease inhibitor cocktail||Life Technologies||78440|
|BCA protein assay||Life Technologies.||23227|
|NuPAGE® Novex® 4–12% Tris-Acetate Protein Gels||Life Technologies||NP0321BOX|
|SuperSignal™ West Femto Maximum Sensitivity Substrate||Life Technologies||34094|
Western Blot Analysis:
- To extract intracellular protein, cells were placed on ice, washed twice with ice-cold PBS and lysed immediately with RIPA buffer (Life Technologies, 89900) supplemented with a protease inhibitor cocktail (Life Technologies, 78440). Notes: Cell lysates need to be sonicated and the soluble fraction was collected after centrifugation.
- Protein amount was quantified by the BCA protein assay (Life Technologies, 23227).
- 20 μg of total protein for each sample was mixed with loading buffer and incubated for 5 min at 95 °C before being loaded into NuPAGE® Novex® 4–12% Tris-Acetate Protein Gels (Life Technologies, NP0321BOX) for electrophoresis.
- Separated proteins were transferred to a PVDF membrane (Millipore, IPSN07852) after electrophoresis. The membrane was blocked with 5% non-fat milk in TBST (50 mM Tris–HCl at pH 8.0, 150 mM NaCl, 0.1% Tween 20) followed by overnight incubation with the primary antibody at 4 ° C.
- A peroxidase conjugated horse anti-mouse IgG secondary antibody (1:20000, Vector Laboratories, PI-2000) or peroxidase conjugated goat antiRabbit IgG secondary antibody (1:20000, Vector Laboratories, PI-1000) was used for western blot.
- ECL signals were developed and detected using the SuperSignal™ West Femto Maximum Sensitivity Substrate (Life Technologies, 34094) and a CDiGit Chemiluminescent Western Blot Scanner (LI-COR).