Cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.5, 0.5% DOC, 0.1% SDS, 1% NP-40, and 150 mM NaCl) with protease and phosphatase inhibitors (#4693159001 and #4906837001; Roche), followed by sonication at the highest setting with a pulse of ± 30 s. After centrifugation at 16,000g for 30 min, lysates were collected in a fresh tube and protein concentration was determined using BCA assay (Pierce). 6× loading buffer was added to lysates, and they were boiled at 95°C for 10 min. Equal amounts of lysates were loaded on 4–20% precast gradient gels (Bio-Rad) and separated at 80–100 V. Proteins were blotted using PVDF membrane (Bio-Rad) and blocked for 1 h at RT. Membranes were incubated overnight with primary antibody followed by washing. For detection, HRP-labeled secondary antibodies (DAKO) followed by incubation with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) were used, or LI-COR Biosciences secondary antibodies (IRDye 680 or IRDye 800) were used followed by detection by Odyssey Imaging Systems or Bio-Rad Laboratories ChemiDoc Imaging Systems.
Free full text: Click here
