JCS -- Ruas et al. 118 (2): 301 Figure IG1

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Fig. 1. CBP mediates the interaction of SRC-1 with HIF-1 ${alpha}$ . (A) Interaction of HIF-1 ${alpha}$ with endogenous CBP is dependent on the integrity of both HIF-1 ${alpha}$ transactivation domains. Cells were transfected with 1 µg pFlag-mHIF-1 ${alpha}$ (Flag-HIF-1 ${alpha}$ ) or pFlag-mHIF1 ${alpha}$ (532 ${Delta}$ 583)(L808A/L809A) [Flag-HIF-1 ${alpha}$ (532 ${Delta}$ 583)LL808AA] and kept at normoxia (N) or treated with 100 µM CoCl₂ (C) for 3 hours. 3 mg protein from HEK 293 whole cell extracts was incubated with anti-CBP antibody ( ${alpha}$ -CBP), and precipitated complexes were separated by SDS-PAGE followed by immunoblotting using anti-CBP and anti-Flag ( ${alpha}$ -Flag) antibodies. (B) CBP immunodepletion abolishes interaction between HIF-1 ${alpha}$ and SRC-1. HEK 293 cells were transfected with 1 µg of pFlag-mHIF-1 ${alpha}$ and kept at normoxia (N) or treated with 100 µM CoCl₂ for 3 hours (C). CBP immunodepletion (CBP ID) was performed by incubating whole cell extracts with either IgG (-) or anti-CBP antibodies (+) for 2 hours at room temperature. Following immunodepletion whole cell extracts were recovered and incubated with anti-SRC-1 antibody ( ${alpha}$ -SRC-1) for an additional 2 hours. Precipitated complexes were separated by SDS-PAGE and analyzed by immunoblotting using anti-Flag, anti-CBP and anti-SRC-1 antibodies. ^*Non-specific band. Input gels were loaded with 50 µg protein of each whole cell extract.