The WBSCR22 protein level is regulated by ubiquitin-proteasome pathway.

(A) Flow cytometric analysis of HeLa cells transfected with plasmids encoding for EGFP, EGFP-WBSCR22, EGFP-WBSCR22-KT/AA and EGFP-WBSCR22-D117A proteins treated with proteasome inhibitor MG132. 24 hours after transfection cells were treated with MG132 for 16 hours, cells were harvested and the amount of EGFP positive cells was analyzed by flow cytometry. (B) Analysis of HeLa cells expressing the WBSCR22 protein treated with MG132 by immunoblotting. For western blot analysis, antibodies against E2Tag, TRMT112 and tubulin were used. (C) Western blot analysis of HeLa cells treated with TRMT112 siRNA and MG132. HeLa cells were transfected with siRNA and MG132 was added 56 hours later. Cells were collected 72 h post-transfection, and analyzed by immunoblotting with antibodies against WBSCR22, TRMT112 and tubulin. (D) WBSCR22 is ubiquitinylated in the cells. HeLa cells were transfected with plasmids encoding E2Tag-WBSCR22, E2Tag-WBSCR22-D117A and HA-Tag-ubiquitin and 24 hours later treated with 10 μM MG132 for 4 hours. Co-immunoprecipitation was performed with anti-E2Tag antibody, the samples were analyzed by immunoblotting using antibodies against WBSCR22 and HA-tag. Proteins of the extract (Input; 3%) and pulled-down fraction (IP) were analyzed by immunoblotting.