Fig. 3

Effect of the PPARγ mutations on RXRα and coactivator interactions. a Representative data set used for SPR (surface plasmon resonance) analysis of the interactions of PPARγ WT or mutants with RXRα showing enhanced interaction between PPARγ T475M and RXRα. Report point 4 s before injection stop was used for the analysis. Equilibrium responses are plotted as a function of total protein concentration and fit to simple 1:1 binding isotherms. Data analysis by 1:1 kinetic model and mean kinetic parameters and equilibrium dissociation constants are reported in Supplementary Fig. 4. b Effect of PPARγ mutations on the PGC1α peptide interaction. The binding affinity of the PGC1α NR1 motif for the purified WT and mutant PPARγ LBDs, as determined by microscale thermophoresis. Unlabeled PPARγ LBD protein was titrated into a fixed concentration of fluorescently labeled PGC1α peptide in the absence of ligand (top left), in the presence of three equivalents of rosiglitazone (top right) or of three equivalents of GW1929 (bottom left). Isotherms averaged over three consecutive measurements and fitted according to the law of mass action to yield the apparent K_d. Each plot is representative of at least two independent experiments performed with different batches of protein preparation. c Mammalian two-hybrid analysis reveals increased interaction of PPARγ mutants (M280I, I290M and T475M) with MED1 coactivator domain. pG5-Firefly luciferase reporter plasmid was co-expressed with VP16-PPARG (WT or mutants) and with GAL4-DNA-binding domain-fused MED1. Renilla luciferase, expressed under the control of the CMV promoter, was used to normalize the signal. The data shown are the means ± SD of one representative experiment conducted in quadruplicate. Results (means ± SD) of three independent experiments are also provided as Supplementary Fig. 16. The results for each mutant were compared with those for the WT in Dunnett’s multiple comparisons test, *0.01 < p < 0.05; **0.001 < p < 0.01. a–c Source data are provided as a Source Data file