Fig. 8. p62 and NBR1 regulate PPARγ-RXRα heterodimerization to control thermogenesis in brown adipocytes.

a FLAG-RXRα, GST-PPARγ, WT/mutants of HA-p62, and HA-NBR1 were overexpressed in HEK293T cells and the interaction of RXRα with PPARγ was analyzed by immunoblotting in pull-downs using glutathione-beads against GST-PPARγ, in the present of NBR1 and/or p62. Representative immunoblotting and densitometric quantification were shown (n = 3 independent experiments). EV empty vector, wt wild-type, mu mutant. b Endogenous interaction of PPARγ with RXRα in BAT of mice exposed to cold for 7 h. PPARγ immunoprecipitates were analyzed by immunoblotting. Densitometric quantification was shown (n = 6, per Sqstm1^f/f, Sqstm1^AKO, Sqstm1^f/fNbr1^f/f, and Sqstm1^AKONbr1^AKO). c Endogenous interaction of PPARγ with RXRα in double KO iBAs (sgSqstm1sgNbr1) reconstituting p62, NBR1, or both. PPARγ immunoprecipitates were analyzed by immunoblotting. Densitometric quantification was shown (n = 3 independent experiments). d Luciferase assay determining transcriptional activity of PPARγ in iBAs transfected with indicated cDNA vectors, cells were treated with ISO (1 µM) and rosiglitazone (1 µM) for 48 h (n = 6 biological replicates). e qPCR analysis of Ucp1 expression in Sqstm1^–/–Nbr1^–/– iBAs reconstituting p62 or NBR1 or both with/without overexpression of PPARγ. Cells were treated with ISO (1 µM) for 48 h. EV (n = 3 biological replicates), PPARγ (n = 5 biological replicates). Data are presented as mean ± SEM (a–e). *p < 0.05, **p < 0.01. Two-tailed Student’s T-test (a–e). Source data are provided as a Source Data file.