Co-immunoprecipitation and ChIP-reChIP of FOXAs reveals interaction and co-binding among FOXAs. (a) Immunoprecipitations were performed with indicated antibodies on nuclear extracts of HepG2 cells and the immunocomplexes were detected with FOXA1, FOXA2, and FOXA3 antibodies. IP, immunoprecipitation; IgG, the antibody was replaced by normal IgG; Nuc, total nuclear extract; ID, immunodepleted fraction obtained after IP. The blots are representative of two or three replicates. None of the proteins was overexpressed. (b) ChIP-reChIP of FOXA1, FOXA2, and FOXA3 tested by semiquantitative PCR. The order of antibodies used to immunoprecipitate the protein-DNA complex is indicated to the left. In each pair of bands, the left one is for the IP and the right for input. Pairs 1, 5, and 9: a primer amplifying a region with binding site for both proteins; pairs 2 and 6: regions with binding sites for FOXA1, but not FOXA2 or FOXA3, respectively; pairs 3 and 10: regions with binding sites for FOXA2, but not FOXA1 or FOXA3, respectively; pairs 7 and 11: regions with binding sites for FOXA3, but not FOXA1 or FOXA2, respectively; pairs 4, 8, and 12: a region with no FOXA binding. FOXA2-FOXA1, FOXA3-FOXA1, and FOXA3-FOXA2 were performed as independent experiments from the other three ChIP-reChIPs.