Figure 6.
Affixin249–272 specifically inhibits α-actinin–affixin interaction. (A) The minimum α-actinin–binding site of affixin was coimmunoprecipitated with α-actinin. The expression vector encoding the flag-tagged α-actinin was cotransfected into CHO-K1 cells with pEGFP-affixin249–272. Immunoprecipitation assay was performed using the anti-flag antibody, and immunocomplexes were subjected to immunoblot analysis with monoclonal anti-flag and anti-GFP antibodies. (B) Endogenous affixin was immunoprecipitated from lysates of CHO-K1 cells harvested from fully spreading cells transfected with GFP-affixin249–272 or GFP vector alone and cultured for 24 h. Affixin immunoprecipitates were analyzed by Western blotting with anti-α-actinin, affixin, GFP, ILK, paxillin, vinculin, and actin antibodies as indicated. Note that the coimmunoprecipitation of α-actinin, but not ILK, was abolished in GFP-affixin249–272–expressing cells.