Fig. 5
TRIM22 ubiquitinastes p21 and promotes K63-linked ubiquitination of p21. (A) GeneMANIA online software was used to analyze the interaction between TRIM22 and p21. (B) HEK293T cells were transfected with p21-SF and Myc-TRIM22, the cell lysates were immunoprecipitated with anti-Myc and anti-Flag antibodies, respectively. Then, anti-Myc and anti-Flag antibodies were used to detect the immunoprecipitates, and exogenous interactions of TRIM22 and p21 were tested by immunoblotting. (C) HEK293T cells were transfected with SF-TRIM22, the cell lysates were immunoprecipitated with anti-p21 and anti-Flag antibodies, and endogenous interactions of TRIM22 and p21 were tested by immunoblotting. (D) SF-tag full-length and truncated TRIM22 constructs were transfected into HEK293T cells, lysates were pulled-down with S-protein agarose to elucidate the interacting domain of TRIM22 to p21. The schematic representation of TRIM22 deletion mutants(up), and the interaction between p21 and TRIM22 truncations (below). (E) Ubiquitination assay of p21 in HEK293T cells co-transfected with p21-SF, MYC-TRIM22, and UB-HA, and the transfected 293T cells were treated with MG132 before proteins were harvested. Then the cells were lysed and pulled-down with S-protein agarose, and the indicated protein levels were tested by immunoblotting. (F) Ubiquitination assay of p21 in HEK293T cells co-transfected with Myc-TRIM22, p21-SF, UbHA, or K27R-HA, K48R-HA, and K63R-HA with the treatment of MG132. Then the cells were lysed and pulled-down with S-protein agarose, and the indicated protein levels were tested by immunoblotting.
