Iblot dry blotting gel transfer system

Cells were washed with ice-cold PBS and lysed with RIPA buffer supplemented with 2-mercaptoethanol (final concentration 6%) and Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), then boiled for 5min at 95°C. Equal amounts (10-30 μg) of samples were loaded and proteins were separated by SDS-PAGE using 4-12 % Bis-Tris gels (Bio-Rad, Hercules, CA, USA), followed by transfer to PVD membranes using iBlot Dry Blotting Gel Transfer System (Thermo Fisher Scientific). Membranes were blocked for 1 h in 5% BSA-TBST at room temperature, and then incubated with primary antibodies overnight at 4°C. Blots were washed and incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature, and detection was performed using enhanced chemiluminescence. Primary antibodies were against p16 (Abcam, #ab108349, 1:500 dilution) and p21 (Cell Signaling Technology, #2947s, 1:1000 dilution). Secondary antibodies were HRP-conjugated goat anti-rabbit (Bio-Rad) and goat anti-mouse (Bio-Rad) antibodies. An antibody against beta actin (Sigma, #A2228, 1:10,000 dilution) was used for loading control.

Cells were rinsed with ice-cold PBS and then lysed in RIPA buffer supplemented with 6% of 2-mercaptoethanol and Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). The lysates were subsequently boiled at 95°C for 5 min. Equal amounts (20 μg) of the lysate samples were loaded onto 4–12% Bis-Tris gels (Bio-Rad, Hercules, CA, USA) for SDS-PAGE separation. The proteins were then transferred to PVDF membranes using the iBlot Dry Blotting Gel Transfer System (Thermo Fisher Scientific). Following transfer, the membranes were blocked for 1 h at room temperature in 5% BSA-TBST and then incubated with primary antibodies overnight at 4°C. After washing, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and visualized using enhanced chemiluminescence. The primary antibodies were p16 (Abcam, #ab108349, anti-rabbit, 1:500 dilution). The secondary antibodies were HRP-conjugated goat anti-rabbit and goat anti-mouse (Bio-Rad). Beta-actin (Sigma, #A2228, anti-mouse, 1:10,000 dilution) was used as an internal control.