FIG 3.

Effects of AF2 domain mutations and ligands on physical interactions of PPARγ and RXRα with LCoR. (A) Extracts of 293T cells cotransfected with N-terminally Flag-tagged LCoR and the indicated RXRα and/or PPARγ mutant combinations were resolved without IP (INPUT) or after IP with anti-Flag Ab beads (IP) and probed using Abs against Flag, RXRα, and the N or C terminus of PPARγ; the last two were used to detect PPARγ species lacking the opposite termini. (B) CV1 cells were grown in stripped serum and cotransfected with full-length (FL) RXRα alone (lane 1) or with Flag-LCoR and either FL-RXRα (lanes 2 and 3) or RXRα-ΔAF2 (lanes 4 and 5) in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of LG268. Extracts were resolved without IP (Input) or after IP with anti-Flag Ab beads (top and bottom) and probed with anti-Flag (top) or anti-RXRα (middle, bottom) Abs. (C) TSC (gy11, WT, lanes 1 to 5, and gy9, Pparg null, lane 6) were cultured in the presence (lane 1) or absence (lanes 2 to 6) of fibroblast growth factor 4 (FGF4), heparin, and conditioned medium, along with combinations of Rosi and LG268, as indicated. After 4 days of differentiation, nuclear extracts were resolved without IP (Input, 5 μg) or after IP with anti-PPARγ Ab in the presence of the respective ligands (280 μg, top and bottom). Blots were probed with anti-PPARγ (top) or anti-LCoR (middle and bottom) Ab.