The ratio of MuRF2 to PPARγ1 determines the ubiquitin post-translational modification and ligand-dependent protein levels. a Immunoblot analysis of cardiac LV PPARα, PPARβ, and PPARγ1 levels normalized to GAPDH. N = 3/group. b Increasing MuRF2 results in a PPARγ1 ligand (Rosiglitazone)-dependent decrease in PPARγ1 in vitro 24 h after transfection. HEK293 cells were co-transfected with MuRF2 and PPARγ1 (as indicated below graph). After 24 h, 1 microM Rosiglitazone was added overnight and cells harvest at 48 h. *p < 0.05 vs. MuRF2:PPARγ1 ratio of 1:1 without Rosiglitazone. c Immunoprecipitation studies identifying MuRF2 interaction with PPARg1. HEK293 cells were transfected p3XFlag-PPARγ1 (or p3XFlag-Empty vector), pcDNA3.1-HA-MuRF2p50a (or HA-MURF2∆Ring) and immunoblotted for MuRF2 (anti-HA). d
Left Proteasome inhibition with MG132 prevents MuRF2’s degradation of PPARγ1 in a Right ligase-dependent (Ring Finger-dependent) manner. HEK293 cells transfected with p3XFlag-PPARγ1, pcDNA3.1-HA-MuRF2p50a and treated with MG132 (50 μM) for 2.5 h before Rosiglitazone added (1 μm). e–g In vitro ubiquitination assays of MuRF2’s ability to ubiquitinate PPARα (e), PPARβ (f), and PPARγ1 (g), with all lanes having Ub, E1, E2, MuRF2, and PPAR (=full reaction), unless otherwise indicated. Immunoblot for MuRF2 illustrates auto-ubiquitination (=MuRF2 activity) present in the same reaction as mono-ubiquitination (PPARα) and poly-ubiquitination (PPARγ1). Values expressed as Mean ± SE of three independent experiments. A one-way ANOVA was performed to determine significance, followed by a Holm-Sidak pairwise comparison to determine significance between groups. #p < 0.05, **p < 0.01.