Additional file 2: of FBXO22 promotes the development of hepatocellular carcinoma by regulating the ubiquitination and degradation of p21

Figure S2. FBX022 ubiquitinates p21 and F-box domain mediates the process (a) LM3 cells were treated with CHX (10 μM), collected at the indicated time points, and immunoblotted for FBXO22, p21 and GAPDH. Quantification of the p21 levels relative to GAPDH expression is shown. (b and c) HepG2 and LM3 cells were treated with Mg132 (10 μg/ml) for 4 h, total protein was extracted and subjected to western blotting using anti-FBXO22, anti-p21, or anti-GAPDH antibodies. (d and e) HepG2 and LM3 were treated with Mg132 (20 μg/ml) for 4 h, then lysed with IP lysis/wash buffer with protease inhibitor, phosphatase inhibitor and 10 μM N-ethylmaleimide. p21 was immunoprecipitated with an anti-p21 antibody, and the immune-precipitates were probed with anti-FBXO22, anti-ubiquitin and anti-p21 antibodies. (f) schematic representation of the domain structure of FBXO22 (JPG 608 kb)