Figure 8. TRIM23 interacts with PPARγ2 and promotes ubiquitination of PPARγ2.
(A) In vivo assay for interaction between TRIM23 and PPARγ2. FLAG-TRIM23 and HA-PPARγ2 were transfected into HEK293T cells. Lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-FLAG and anti-HA antibodies. (B) In vivo assay for interaction between TRIM23 and PPARγ2 with or without troglitazone (1 μM). FLAG-TRIM23 and HA-PPARγ2 were transfected into HEK293T cells. Coimmunoprecipitation assays were performed using the cell extract from these cells with anti-FLAG antibody in the presence or absence of 1 μM troglitazone. (C) In vivo assay for interaction between TRIM23 and PPARγ2. 3T3-L1 cells stably expressing FLAG-TRIM23 were generated, differentiated, and harvested at day 6. Whole cell lysates were immunoprecipitated with anti-FLAG antibody and then immunoblot analysis was performed with anti-FLAG and anti-PPARγ antibodies. (D) In vivo assay for ubiquitination of PPARγ2 by TRIM23. FLAG-PPARγ2, Myc-TRIM23, and HA-ubiquitin (HA-Ub) were transfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-FLAG antibody and then immunoblot analysis was performed to detect ubiquitination of PPARγ2. (E) Promotion of in vitro PPARγ2 polyubiquitination by TRIM23. An in vitro ubiquitination assay was performed with the indicated combinations of ATP, HA-Ub, His6-E1, His6-E2 (UbcH5B), His6-GST-TRIM23-FLAG, and GST-PPARγ2. Reaction mixtures were subjected to immunoblot analysis with anti-PPARγ (top), anti-HA (middle) or anti-FLAG (bottom). The positions of GST-PPARγ2 or His6-GST-TRIM23-FLAG modified by various numbers of HA-Ub moieties are indicated. (F) Schematic representation of TRIM23 deletion mutants is shown. Protein motifs are indicated. RING, ring-finger domain; B-box, B-box domain; CC, coiled-coil domain; ARF, ADP ribosylation factor domain. (G) Immunoblot analysis of ectopic expression of TRIM23 deletion mutants in TRIM23-knockdown 3T3-L1 cells before induction of adipogenesis. (H) Cells were stained with Oil Red O to visualize the accumulation of lipid droplets at day 6.