Nitrocellulose western blotting membranes

Cells were lysed with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 25 mM Tris with protease inhibitor cocktail (Roche)) and denatured for 5 min in 5 × SDS-PAGE loading buffer. Concentrations of the proteins were determined using the Bradford assay (Bio-Rad) and separated with SDS-PAGE and then transferred to nitrocellulose western blotting membranes (GE Healthcare, 10600001). The membranes were blocked in 5% skim milk powder in Tris-buffered saline-Tween (TBS-T) for 1.5 h at 37 °C, washed with PBS containing 1% Tween-20 (PBS-T), and then incubated with the primary antibody overnight at 4 °C. After washing, the membranes were incubated with the corresponding secondary antibody (conjugated with HRP) at room temperature for 1.5 h and detected with enhanced chemiluminescence (ECL) (Thermo Fisher Scientific, 34580).

Cells were washed with cold PBS and lysed in radioimmunoprecipitation lysis buffer (Thermo Fisher Scientific; catalog no.: 89901) containing a protease inhibitor cocktail (Bimake; catalog no.: B14001) and phosphatase inhibitor cocktail (Bimake; catalog no.: B15001). Lysates were denatured for 10 min in 5× SDS-PAGE sample loading buffer, separated by SDS-PAGE, and transferred to nitrocellulose Western blotting membranes (GE Healthcare; catalog no.: 10600001). Subsequently, the membrane was washed and incubated with PBS containing 0.2% Tween-20 (Sigma–Aldrich; catalog no.: P1379) and 5% nonfat dry milk (BD; catalog no.: 232100), incubated with the primary antibodies at room temperature, followed by horseradish peroxidase–conjugated secondary antibodies (Proteintech Group; catalog no.: SA00001-1), and detected with enhanced chemiluminescence (Share-bio; catalog no.: SB-WB012).