FA proteins interact with HES1. (A) Yeast-2-hybrid assay with HES1 and FA proteins. Yeast strain AH109 was cotransformed with HES1 constructs expressing full-length or truncated HES1 protein with FA proteins as indicated and assayed for interaction as described in “Yeast-2-hybrid.” A positive interaction is indicated as +. Negative controls included pGBK-HES1 cotransformed with pGADT7 empty vector. (B) FA core complex components coimmunoprecipitate with HES1. 293T cells were cotransfected with HA-tagged HES1 and FA coding vectors and were subjected to immunoprecipitation (IP) with either anti-FANCA antibodies, anti-HES1 antibodies, or control IgG. IP was analyzed by SDS-PAGE and Western blotting using the indicated antibodies. (C) Co-IP of endogenous proteins from 293T cell extracts using anti-HES1 or anti-FANCA antibodies. (D) Coomassie gel staining of endogenous protein extracts of 293T cells subjected to IP using anti-HES1 antibodies. Major bands were extracted from gel slices and the indicated proteins were identified by mass spectrometry.