Figure 5.

TRIM32 ubiquitinates OTULIN. (A) HEK293 cells were transfected with TRIM32 or the RING deletion mutant with the NF-κB luciferase reporter and pRL-SV40. After 48 h, luciferase activities were measured. The right panel shows protein expression levels of TRIM32 and the mutant by western blotting. (B) HEK293 cells were transfected with TRIM32 or the C44S/C47S mutant with the NF-κB luciferase reporter and pRL-SV40. After 48 h, luciferase activities were measured. Data represent mean ± SD of three independent experiments. The P-value was calculated (two-tailed Student’s t-test). *P < 0.05. The lower panel shows protein expression levels of TRIM32 and the mutant by western blotting. (C) HEK293 cells were stimulated with 10 ng/ml TNFα for the designated times. Cell lysates were immunoprecipitated with anti-OTULIN antibody and blotted as indicated. (D) OTULIN-FLAG was co-transfected with vector, TRIM32-HA, or the indicated mutant into HEK293 cells. Cell lysates were immunoprecipitated with anti-FLAG antibody and blotted with the indicated antibodies. (E) In vitro ubiquitination of OTULIN by TRIM32. FLAG-tagged OTULIN, HA-tagged TRIM32, or the indicated mutant, plus E1, E2 (UBCH5A), ATP, and ubiquitin were added as indicated and incubated at 30°C for 2 h. The reaction mixtures were immunoprecipitated with anti-FLAG antibody. After washing with 1 M urea, the OTULIN protein was eluted with FLAG peptide. The eluates were blotted with the indicated antibodies. (F) In vitro ubiquitination of OTULIN by TRIM32 and the NHL mutants. HA-tagged OTULIN, FLAG-tagged TRIM32, or the indicated mutant, plus E1, E2 (UBCH5A), ATP, and ubiquitin were added as indicated and incubated at 30°C for 2 h. The reaction mixtures were immunoprecipitated with anti-FLAG antibody. After washing with 1 M urea, the OTULIN protein was eluted with FLAG peptide. The eluates were blotted with the indicated antibodies.