Fig. 6

TRIM56 stabilizes cIAP1 via deubiquitylation. a-b Western blot of lysates prepared from GBM#P3-, LN229- and U118MG-cIAP1^OE cells transfected with Flag-TRIM56 and treated with MG132 (20 μg/mL) for 6 h before collection. Immunoblotting was then performed using K48/63-linked Ub and Flag antibodies. c Western blot analysis performed on extracts prepared from GBM#P3, LN229 and U118MG cells transfected with Myc-cIAP1, Flag-TRIM56 and HA-Ub (as indicated) and treated with MG132 (20 μg/mL) for 6 h prior to collection with indicated antibodies. d Western blot of co-IPs performed on extracts prepared from the stably transfected GBM#P3, LN229 and U118MG cells shown and pulled down with cIAP1 antibody. Blots were incubated with the antibodies indicated including K48-linked specific polyubiquitin antibody. e Schematic representation of the functional domains contained in wild-type TRIM56 and each deletion mutant and western blot of lysates prepared from GBM#P3 cells, LN229 cells and U118MG cells transfected with the different TRIM56 full length and mutant plasmids. Blots were incubated with the antibodies shown. f Western blot of co-IPs performed on extracts prepared from GBM#P3, LN229 and U118MG cells transfected with Myc-cIAP1, HA-Ub and full length or deletion mutants of Flag-TRIM56 as indicated and treated with MG132 (20 μg/mL) for 6 h prior to collection. cIAP1 antibody was used in the immunoprecipitations. g Western blot of co-IPs performed on lysates prepared from cells transfected with Myc-cIAP1 and full length or deletion mutants of Flag-TRIM56 in GBM#P3, LN229 and U118MG cells to determine the interaction between TRIM56 structural domains and cIAP1. Cell lysates were immunoprecipitated with anti-Flag. Comparisons between two independent samples and among multiple samples were performed using two-tailed t tests and one-way ANOVA, respectively. Error bars indicate at least three independent experiments, and data are shown as mean ± SEM. ∗ p < 0.05, ∗  ∗ p < 0.01, ∗  ∗  ∗ p < 0.001, and ∗  ∗  ∗  ∗ p < 0.0001