Trans blot turbo mini or midi nitrocellulose membranes

Protein lysates of cells and tissue were prepared using RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS, 2 mM EDTA) complemented with protease and phosphatase inhibitors (Roche) and protein concentration was quantified using the BCA protein assay kit (Pierce). Protein lysate was loaded onto NuPAGE 4–12% Bis-Tris gradient gels (Invitrogen) and transferred onto Trans-Blot® Turbo™ Mini or Midi Nitrocellulose membranes (BioRad) using Trans-Blot Turbo Transfer System (BioRad). Membranes were blocked in 10% Western Blot Blocking Reagent (Roche) or 3% BSA for 1 h at room temperature (RT). Primary antibody incubation was performed overnight at 4°C. Membranes were washed using TBS-T and subjected to secondary fluorochrome-conjugated antibodies for 1 h at RT and protein was detected using the Odyssey CLx imaging system and processed using ImageJ software 1.48v. Antibodies are listed in Supplemental Table 1.