Western Blotting (WB) and Protein Gel Staining

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology 9806S) containing protease and phosphatase inhibitor (ThermoFisher A32961) and lysates were sonicated and centrifuged (13,200 rpm at 4°C for 20 min). The samples were resuspended in Sodium dodecyl-sulfate (SDS) denaturing buffer (1x Laemmli sample buffer, 5% beta mercaptoethanol, 2.05% SDS), and loaded onto SDS- polyacrylamide gel electrophoresis gels. The separated proteins in the gel were transferred onto nitrocellulose membranes at 40 volts at 60°C for 1.5 h. The membrane was blocked with 5% non-fat milk in Tris buffered saline solution for 1 hour. Primary Ab were diluted 1:1000 and incubated overnight, and secondary ab were diluted 1:3000 or 1:5000 and incubated for one hour. Luminescence detection was performed by ECL (Immobilon Crescendo Western HRP Substrate, MILLPORE WBLUR0500), on a Syngene image analyser (G:BOX). For protein gel staining, protein-containing SDS-PAGE gels were stained with Coomassie Protein Assay Reagent (Thermo scientific 1856209).