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. 2003 Jan;23(1):140–149. doi: 10.1128/MCB.23.1.140-149.2003

FIG. 2.

FIG. 2.

Confirmation of the identity of the components of ASCOM. (A) HiTrap Q column fractions subjected to Western analysis with the indicated antibodies against ASC-2, CBP, SRC-1, and hMed6. FT, flowthrough; fr#, fraction number. (B) HiTrap Q fractions 40 to 44 were pooled and immunoprecipitated (IP) with anti-ASC-2 (αASC-2) antibody and Western (W) analyzed with the indicated antibodies. αCBP, anti-CBP; αSRC-1, anti-SRC-1; αSRC-2, anti-SRC-2. (C) Antibodies were generated against synthetic or recombinant polypeptides encoded by cDNAs isolated based on the MALDI-TOF mass spectrometry data of the purified proteins. + and −, HiTrap Q fractions containing ASCOM from HeLa nuclear extract (fractions 40 to 44) and unrelated fractions (30 to 34), respectively. Each antibody recognized a protein with the expected molecular weight (MW) (in thousands). αALR, anti-ALR; αHALR, anti-HALR; αASH2, anti-ASH2; αRBQ-3, anti-RBQ-3. (D, E, and F) HiTrap Q fractions of HeLa nuclear extract containing ASCOM (fractions 40 to 44) were immunoprecipitated with the indicated antibodies (IP), separated by SDS-4 to 6.5% PAGE, and probed with the indicated antibodies (W). αHA, anti-HA; αBRG1, anti-BRG1. S and s indicate supernatant, and P and p indicate precipitate. Ten percent of the total reaction mixture was loaded as input.