Fig. 4: ARIH2 interacts with p21 and regulates the expression of p21 via ubiquitination in an E3 ligase activity dependent manner.

A Flag-ARIH2 and MYC-p21 plasmids were cotransfected into 293FT cells, and the total cell lysates were immunoprecipitated with anti-Flag and anti-MYC antibodies, respectively. Then, anti-MYC and anti-Flag antibodies were used to detect the immunoprecipitates, and anti-Flag and anti-MYC antibodies were used to test the success of the experiment. B SGC7901 cell lysates were subjected to immunoprecipitation with control IgG and ARIH2 antibodies. The immunoprecipitate was then probed with p21 antibody, and the ARIH2 antibody was used to detect the success of the experiment. C Cell lysates were prepared from the control and ARIH2-overexpressing GC cells that had been treated with MG132 for 6 h. Equal amounts of cell lysates were immunoblotted with the indicated antibodies. D, E The control and ARIH2-knockdown GC cells were treated with CHX (100 μg/ml) for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. Quantification of the turnover of p21 in the control and ARIH2-knockdown GC cells. F Flag-ARIH2, MYC-p21 and HA-ubiquitin plasmids were transiently transfected into 293FT cells for 2 d, and the transfected 293FT cells were treated with MG132 for 7 h before proteins were harvested. The ubiquitinated p21 proteins were pulled down with an anti-MYC antibody and immunoblotted with an anti-HA antibody. G HA-ubiquitin mutants in which only one lysine residue was mutated to an arginine residue, as shown, were used, and co-IP was performed. H MYC-p21 mutants in which only one lysine residue was mutated to an arginine residue, as shown, were used, and co-IP was performed.