Chicken FANCD2 L234R is defective in FANCI binding.

<p>(A, B) Gel filtration analysis of the ID complex formation. cFANCD2, cFANCI, and the mixture of cFANCD2 and cFANCI were fractionated on the Superdex 200 gel filtration column. The SDS-PAGE analysis of the mixture of the cFANCD2 and FANCI fractions is shown below the gel filtration profiles. The void volume of the Superdex200 column is indicated as ‘Vo’ on the gel filtration profiles. Experiments with wild type (WT) cFANCD2 and WT cFANCI (A), cFANCD2 L234R and WT cFANCI (B). (C) Immunoprecipitation analysis of the cFANCD2 and cFANCI interaction. The cell extracts from chicken DT40 cells expressing GFP-WT cFANCD2 or cFANCD2 L234R were immunoprecipitated with an anti-GFP antibody. The immunoprecipitates were separated by 6% SDS-PAGE, and cFANCD2 and cFANCI were detected by western blotting using anti-chicken FANCD2 and FANCI antibodies, respectively. (D) Graphic representation of the relative intensity of the bands corresponding to GFP-cFANCD2 (upper) and endogenous cFANCI (lower) shown in panel (C). The band intensity was normalized relative to that of cFANCD2 or cFANCI.