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. 2001 Nov;21(22):7617–7628. doi: 10.1128/MCB.21.22.7617-7628.2001

FIG. 3.

FIG. 3

A CA150-interacting protein of 80 kDa (CIP80), which interacts directly with CA150, is the splicing-transcription factor SF1. (A) Diagram showing the recombinant CA150 proteins used as probes in the far-Western analysis. Abbreviations are the same as those used in Fig. 1. N-CA150, WW2mt, and WW3mt contain amino acids 235 to 631 of CA150, except that the WW2mt probe had the central aromatic residues of WW2 domain mutated from YYY to AAA and WW3mt had the corresponding residues mutated from FFY to AAA. These mutations ablate the recognition of proline-containing ligands by the WW domains. (B) Far-Western analysis of HeLa nuclear extract with the N-CA150 probe, as indicated at the top of the figure, detected a CA150-interacting protein with an apparent mass of 80 kDa, designated CIP80. The negative-control far-Western blot with a GST probe did not bind to nuclear proteins, as expected. (C) Far-Western analysis of HeLa nuclear extract showed that mutation of the WW2 domain ablates binding to CIP80 while mutation of the WW3 domain has no effect. CA150 probes are indicated at the top, and CIP80 is indicated on the left. (D) Coimmunoprecipitation of CIP80 with CA150 from HeLa nuclear extract. CA150 was immunoprecipitated using antigen affinity-purified rabbit polyclonal antibodies, as indicated at the top. The immunoprecipitates were analyzed by Western blotting with anti-CA150 antibodies (top panel) and by far-Western blotting using the N-CA150 probe (bottom panel), as labeled on the left. (E) Purification scheme for isolating CIP80. The CIP80 protein was excised from an SDS-PAGE gel and microsequenced by mass spectrometry. CIP80 corresponds to the previously identified SF1 protein. (F) Fractions from HighS chromatography, developed with a linear gradient of 100 to 500 mM KCl, were analyzed by far-Western blotting with the N-CA150 probe. Molecular weight markers are indicated on the right of the figure in thousands. CIP80 is labeled on the left. The peak CIP80-containing fraction from the phosphocellulose step was included in the analysis and labeled Input. A 110-kDa protein that also interacts with the N-CA150 probe, albeit weakly, copurified with CIP80.