Fig. 6: TRIM31 interacts with RHBDF2 and mediates degradation of Rhbdf2 by K48-linked polyubiquitination.
a Representative western blotting for co-immunoprecipitation (CO-IP) detection in L02 cells transfected with HA- or Flag-tagged RHBDF2 or TRIM31 vectors. The anti-Flag or anti-HA antibodies were used as probes (n = 4 per experiment). b The glutathione S-transferase (GST) pull-down assays in which either GST-tagged RHBDF2 (RHBDF2-HA-GST) or blank HA-GST was used to pull down TRIM31-Flag or GST-tagged TRIM31 (TRIM31-HA-GST) or blank HA-GST was used to pull down RHBDF2-Flag. Purified GST was used as the control (n = 4 per experiment). c The binding domains of RHBDF2 and TRIM31 were detected using full-length and truncated Rhbdf2 or Trim31 expression vectors based on IP assays. Anti-Flag or anti-HA antibodies were used to confirm the binding sites of RHBDF2 and TRIM31, respectively (n = 4 per experiment). d The ubiquitination levels of Rhbdf2 in the liver samples of Trim31-Flox or THKO mice in the presence of HFD feeding for 16 weeks (n = 3 mice per group). e Representative western blotting assays of lysates from L02 cells transfected with Myc-tagged ubiquitin (Myc-Ub), RHBDF2-HA, full-length TRIM31-Flag, and TRIM31 with RING domain mutant (TRIM31-Flag RINGΔ), followed by IP with anti-HA, probed with K48-Ub or anti-Myc. f Representative western blotting assays of lysates from 500 μM PA-treated WT L02 (WT-L02) cells or L02 with TRIM31 knockout (THKO-L02) cells for 4 h, followed by IP with anti-RHBDF2, probed with anti-Ub, K48-Ub, K63-Ub. g Ubiquitination levels of RHBDF2 after TRIM31-Flag overexpression and in response to PA administration in L02 cells co-transfected with RHBDF2-HA and the indicated Myc-tagged ubiquitin constructs (K48O, K63O, K33O, K6O, K29O, K27O) (upper). K48O means ubiquitin in which all lysines except K48 were mutated. The empty vector was used as a control. Representative western blotting indicating the ubiquitination levels of RHBDF2 in L02 cells transfected with the K48O vector in different combinations and the indicated downstream events cascades protein expression levels in WCL (lower). h Representative images of the intracellular TG levels in THKO-L02 cells transfected with adenovirus-packed full-length TRIM31 sequences (AdTRIM31) or different truncated TRIM31 sequences, which were then incubated with 500 μM PA for 4 h. The adenovirus-containing GFP vector (AdGFP) was used as controls (magnification, ×100; n = 10 images per group for each staining) (##P < 0.01 vs. AdTRIM31 groups; **P < 0.01 vs. AdGFP groups). The relevant experiments presented in this part were performed independently at least three times. Data are expressed as mean ± SEM. The relevant experiments presented in this part were performed independently at least three times. Significance determined by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test.
