Fig. 4: TRIM22 ubiquitinates IκBα.
From: TRIM22 activates NF-κB signaling in glioblastoma by accelerating the degradation of IκBα
a Luciferase activity for NF-κB luciferase or control reporter constructs in modified LN229 and U118MG cells. b Western blot analysis of cytoplasmic (Cyto) and nuclear (Nuc) fractions prepared from indicated cells. Immunofluorescence for P65 in modified LN229 and U118MG cells showing cellular localization. Scale bars, 20 μm. c Western blot to detect IκBα levels after 0, 4, 8, and 12 h of cycloheximide (CHX; 25 μg/mL) treatment in modified LN229 and U118MG cells compared with controls. d Line graph showing IκBα protein levels normalized to β-tubulin and to 0 h at the indicated time points. e Western blot of IP of IκBα incubated with anti-K48-linkage specific polyubiquitin antibody to detect ubiquitination of IκBα in an in vitro assay. f In vivo ubiquitination assay of IκBα. g Western blot analysis of co-IPs performed on lysates prepared from HEK293 cells transfected with Flag-TRIM22 and HA-IκBα. h Western blot analysis of co-IPs performed using anti-IκBα or -TRIM22 antibody on lysates prepared from U87MG, LN229, and U118MG cells. i Schematic representation of wild-type IκBα and the indicated deletion mutants. Western blot analysis of co-IPs performed on lysates prepared from HEK293 cells transfected with Flag-TRIM22 alone or together with indicated HA-IκBα constructs. Upper panels represent co-IPs performed with anti-HA; lower panels represent input protein. j Schematic representation of wild-type TRIM22 and the indicated deletion mutants. Western blot analysis of co-IPs performed on lysates prepared from HEK293 cells transfected with HA-IκBα alone or together with indicated Flag-TRIM22 constructs. Student’s t test: n.s. not significant, *P < 0.05, **P < 0.01.
