Ab112046

The cell culture medium was discarded, and cells were washed with PBS twice. Cells were lysed with RIPA Lysis Buffer (containing protease inhibitor and phosphatase inhibitor) for 20 min, and centrifuged for 20 min at 12,000 rpm and 4°C. The supernatant was aspirated, protein concentration was determined by Bradford assay, and SDS-PAGE was performed. After SDS-PAGE, the protein was transferred to a PVDF membrane. At room temperature, the PVDF membrane was blocked with 5% skim milk for 1 h. Then, the membrane and the primary antibody diluent were blocked overnight. The next day, the membrane was washed with TBST thrice for 10 min each. The secondary antibody was incubated with the membrane for 1 h at room temperature. The membrane was washed with TBST thrice for 10 min each. Then the assay was performed by ECL. The primary antibodies used in this experiment were as follows: TFCP2 (proteintech, 15203-1-AP, 1:1000), tubulin (Santa Cruz Biotechnolog, sc-5286, 1:4000), Flag (Sigma, F9291; 1:3,000), HA(Sigma, H3663, 1:2000), GAPDH (proteintech, 60004-1-Ig, 1:5000), GST (proteintech, 10000-0-AP, 1:3000), P16 (proteintech, 10883-1-AP, 1:1000), P27 (proteintech, 25614-1-AP, 1:1000), P21 (proteintech, 10355-1-AP, 1:1000), SREBP2 Full length (Abcam, ab112046, 1:1000), SREBP2 N-terminal (Abcam, ab30682, 1:1000), HMGCR (Abcam, ab174830, 1:1000).