Fig. 4.

TRIM7 interacts with BRMS1 and ubiquitinates it. (a) Purification of the TRIM7 complex was carried out according to the procedure described in Materials and Methods. Proteins were separated on SDS-PAGE and stained with Coomassie Blue. (b) List of TRIM7-associated proteins identified by MS analysis. (c) HOS and MG63 cell lysates were subjected to IP with anti-TRIM7, anti-BRMS1 or control IgG antibody. (d) The subcellular localization of TRIM7 (red) and BRMS1 (green) was measured by immunofluorescence. Nuclei were stained with DAPI (blue) for reference. Scale bar: 50 μm. (e) qRT-PCR and Western blot analysis showing the effect of TRIM7 overexpression on endogenous BRMS1 levels in SAOS2 cells in the presence of 10 μM proteasome inhibitor (MG132) or DMSO. (f) IP and western blot showing the effect of TRIM7 overexpression on the ubiquitination of BRMS1 in SAOS2 cells. (g) 293T cells were cotransfected with the Flag-BRMS1 (WT) or Flag-mutant BRMS1 constructs (K8R, K69R or K184R) along with myc-TRIM7 and His-Ub constructs, and the pull-down assay was carried out. All the experiments were repeated at least three times, and data are represented as mean ± SD.