FIGURE 4.

Interaction of FBP21 and SIPP1. A, endogenous SIPP1 coimmunoprecipitates with EGFP-FBP21. EGFP and EGFP-FBP21 fusions were transfected into HEK293T cells. After lysis of the cells, FBP21 was immunoprecipitated from the lysates with anti-EGFP antibodies. The coimmunoprecipitated SIPP1s were detected by Western blot analysis with anti-SIPP1 antibodies. The immunoprecipitates were obtained with (+) and without (−) primary anti-EGFP antibodies. The immunoprecipitation of EGFP-FBP21 (variants) is shown in supplemental Fig. S3A. B, interaction of purified, recombinant FBP21 and GST-SIPP1. The interactions in vitro were examined by GST pulldown assay and further detected by Western blot analysis with anti-His (H-3) antibodies, as visualized by the Odyssey Infrared Imaging System. Four GST fusion SIPP1 fragments (1–372, 180–372, 253–641, and 358–641) were examined, the former two fragments contain the first proline-rich region and the latter two contain the second proline-rich region of SIPP1.GST alone was used as a negative control. The expression of GST-SIPP1 fusions is shown in supplemental Fig. S3B and further verified by Western blotting with anti-GST antibodies (supplemental Fig. S3C).