Fig. 3

c-Myc promotes TRIM32 mediated proteasomal degradation of SIRT4. A Western blot analysis of SIRT4 and c-Myc expression in the whole cellular lysates treated with c-Myc shRNA or MG132 for 48 h. B Western blot analysis of TRIM32 and c-Myc expression in whole cellular lysates treated with c-Myc shRNA. C SIRT4 interacts with TRIM32 in vivo. SNU449/HEK293 cell lysates were immunoprecipitated (IP) with control IgG, anti-SIRT4 or anti-TRIM32 antibodies, and then the precipitated proteins were detected by anti-TRIM32 or anti-SIRT4 antibodies, respectively. D SIRT4 degradation curves under various conditions. TRIM32 was knockdown with shRNA or forced overexpressed in SNU449 cells with or without being treated by proteasome inhibitors MG132 or lactacystin. After being treated with protein synthesis inhibitor cycloheximide (CHX; 50 μg/ml), the above cells were collected at the indicated hours. SIRT4 protein levels were detected by immunoblotting in the SNU449 total cell lysates. E SNU449 cells expressing FLAG-SIRT4 and co-expressing with or without hemagglutinin-tagged ubiquitin (HA-Ub) were co-transfected c-Myc and TRIM32 shRNA (left) or co-transfected c-Myc shRNA and TRIM32 (right) for 48 h. After being treated with 5 µM MG132 for 8 h, cells were collected and IP was performed by using FLAG antibody. Polyubiquitination of FLAG-SIRT4 were detected by anti HA-Ub antibody. F The indicated lysine sites of SIRT4 were mutated into arginine. G Lysates from SNU449 cells expressing the above SITR4 site mutants and co-expressing with or without hemagglutinin-tagged ubiquitin (HA-Ub) were pulled down with anti-FLAG, and then immunoblotted with anti HA-Ub antibody. Group differences were analyzed by two-tailed Student’s t test (D) (*p < 0.05, **p < 0.01)