CtBP1 interacts with components of the FA core complex. (A) CtBP1 interacts with FANCA, FANCF, FANCG and FANCL in yeasts. Yeast 2-hybrid assays were performed using FA core complex components as baits and CtBP1^175-440 as prey. Negative and positive interactions are indicated as (−) and (+), respectively. The negative control consisted of a CtBP1^175-440 bait vector with an empty prey vector. Positive controls included p53 bait and SV40 T antigen prey vectors (not shown). Each experiment was performed a minimum of 3 times in triplicate with each gene cloned into either the bait or prey vector (except for FANCG and FANCL). (B) CtBP1 co-immunoprecipitates with FA core complex proteins. HEK293T cells were cotransfected with T7-tagged CtBP1 and plasmids expressing FANCA, FANCC, FANCE, FANCF, FANCG, or FANCL. WCEs were then subjected to IP, followed by SDS-PAGE and IB with the indicated antibodies. Negative IP controls were performed using goat (G) and rabbit (R) IgG. Data shown are representative of 2 identical and separate experiments. (C) Co-IP of endogenous proteins from FANCD2-complemented PD20 cell extracts with anti-CtBP1, anti-FANCA, anti-FANCC, anti-FANCE or anti-FANCD2 antibodies. Negative IP control was done using rabbit IgG (R). Results shown are representative of at least 4 independent experiments performed in different cell lines, including HEK293T, HeLa, FANCA-complemented PD220, or FANCD2-complemented PD20 cells. (D) Confocal immunofluorescence of CtBP1 and FANCA localization in HeLa cells. Cells were double-stained with anti-FANCA (green) and anti-CtBP1 (red) antibodies and were visualized using a Nikon E800 microscope equipped with C1 confocal system at 100× magnification. Shown are 3 representative experiments in which at least 25 cells were analyzed per experiment.