Monoclonal anti gapdh

Western blotting was performed to detect the protein expression levels of CDC42 and CCND1 from tissues and cells, as previously described (13 (link)). Total proteins were extracted with RIPA buffer (Beyotime Institute of Biotechnology). The BCA kit (Beyotime Institute of Biotechnology) was used to determine the concentration. In total, 80 µg of each sample were resolved using 10% SDS-PAGE at 75 V for 2 h. After transfer to PVDF membranes at 350 mA for 2 h, membranes were blocked with 5% skim milk diluted in TBS for 1 h at room temperature. The membranes were probed overnight at 4°C with the following primary antibodies: Monoclonal anti-CDC42 (rabbit; 1:1,000; cat. no. 2466S; Cell Signaling Technology, Inc.), monoclonal anti-CCND1 (rabbit; 1:1,000; cat. no. 3300S; Cell Signaling Technology, Inc.) and monoclonal anti-GAPDH (rabbit; 1:1,000; cat. no. 5174S; Cell Signaling Technology, Inc.; used as an internal control). After probing with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit IgG; 1:5,000; cat. no. TA130059; OriGene Technologies, Inc.) at room temperature for 1 h, proteins were detected using an enhanced chemiluminescence reagent (Bio-Rad Laboratories, Inc.), and an Alpha Innotech instrument (Bio-Rad Laboratories, Inc.) was used to scan and quantify the images. GAPDH was used to normalize the signals.