Ab76253

After transfection for 48 h, RIPA lysis containing proteinase inhibitors (Beyotime Institute of Biotechnology, Haimen, China) and phenylmethylsulfonyl fluoride were used to extract the total protein from HCC cells. The protein concentrations were tested with the BCA protein assay kit (Beyotime Institute of Biotechnology). The total proteins (50 µg) were added into the SDS-PAGE gels and performed electrophoresis at 60 V when bromophenol blue ran out from the bottom. The proteins were then transferred to nitrocellulose filter (NC) membranes. Then, skimmed milk (5-10%) was used to block the proteins on the membranes at room temperature for 2 h. Subsequently, the membranes were incubated with the primary antibody: Rabbit polyclonal anti-DAB2 (ab76253; 1:1,000; Abcam, Cambridge, MA, USA) were added in to incubate the samples at 4°C, and then horseradish peroxidase-conjugated (HRP, 1:10,000). GAPDH primary antibody (5174P; 1:5,000; Cell Signaling Technology, Inc., Danvers, MA, USA) was chosen as the internal reference. After being washed three times with 1X TBST, they were incubated with secondary antibody goat anti-rabbit IgG-HRP (sc-2,004; 1:3,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at room temperature for 2 h. Protein bands were detected using the chemiluminescence method (ECL; Millipore, Billerica, MA, USA).