Fig. 5
FBX022 ubiquitinates p21 via the F-box domain (a and b) HLF and Hep3B 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. (c and d) HLF and Hep3B cells were treated with Mg132 (10 μg/ml) for 4 h, total protein was extracted and subjected to western blotting using anti-Flag, anti-p21, or anti-GAPDH antibodies. (e and f) HLF and Hep3B 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-ubiquitin, anti-FBXO22, anti-p21 and anti-GAPDH antibodies. (g) HEK293T cells transfected with Flag-p21, HA-ubiquitin, Myc-FBX022 and Myc-FBX022ΔF-BOX in combination 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. Flag-p21 was immunoprecipitated with a Flag antibody, and the immune-precipitates were probed with anti-HA, anti-Flag, anti-Myc and anti-GAPDH antibodies