Fig. 2.
Ligands and E. faecalis generate functional effects on DNA-binding PPARγ1 in HT-29 cells. (A) EMSA of pioglitazone (Pio) and GW9662 (GW) stimulation at different time points. Complexes I and II are as indicated. (B) Supershifts (*) by phospho-PPARγ (P-Pγ), total polyclonal PPAR-γ [Pγ (po)], and total monoclonal PPAR-γ [Pγ (mo)], together with control IgG antibodies. (C) Oligonucleotide pulldown of nuclear extracts from cells stimulated by rosiglitazone for 30 min. (D) EMSA with cocultured HT-29 nuclear extracts showing effects of four E. faecalis (all at 106) isolates (EC1, EC3, EC15, and EC16) on DNA-binding property of PPARγ1 at indicated time points. (E) Supershift by phospho-PPARγ (P-Pγ) and control IgG antibodies of nuclear extracts from HT-29 cocultured with EC16. (F) Phospho-PPARγ levels in oligonucleotide pulldown assay of nuclear extracts from HT-29s cocultured with EC16 and control (NT) for varying times as indicated. The PPARγ partner, RXR, is shown to establish the presence of a true receptor complex. The nonspecific DNA-binding protein, Ku70, was shown as a loading control. Data are representative of at least three independent experiments.