Figure 4

Y355X mutation reduces PPARγ protein stability and transcriptional activity. (A) Western blot of lysates from NIH 3T3 cells transfected with the indicated amount of plasmid expressing either FLAG tagged Y355X (fl355X) or wild-type (flWT) PPARγ proteins. PPARγ was visualized with anti-FLAG antibodies and ECFP produced from co-transfected plasmid (visualized with an anti-ECFP antibody) was utilized as a transfection and loading control. The numbers below the panel represent the density of each PPARγ-FLAG band normalized to the ECFP band. Roughly equal amounts of mutant and WT protein expression were achieved by a 15 fold excess of transfected mutant over WT PPARγ DNA. (B) Rosiglitazone dose-response curves for WT and Y355X PPARγ in NIH 3T3 cells transfected with 1 ng of WT or 15 ng of Y355X expression plasmids (non-FLAG-tagged versions) or 15 ng of empty vector, together with a PPAR responsive reporter construct (pFATP-luc) and a β-galactosidase reference plasmid. Data are normalized to the WT vehicle controls and are means ± SD (n = 3).