Fig. 2. Interaction of the mDia1 FH1 domain with profilins. (A) Autoradiograms of supernatants and pellets obtained after affinity precipitation of in vitro translated [35S]-labeled mDia1 protein and its fragments with mouse profilins I and IIa. Profilins were coupled to NHS-HiTrap material, incubated with in vitro translated [35S]-methione-labelled mDia1 polypeptides and subjected to centrifugation. Aliquots of supernatants (S) and pellets (P) were separated by SDS-PAGE, blotted and subjected to autoradiography. Control: BSA coupled to NHS-HiTrap material. Coprecipitation of the in vitro translated products depends on the presence of the FH1 domain, as in wt mDia1 and 456-761mDia1. (B) Immunoblots obtained by SDS-PAGE from immunoprecipitates of profilin I from extracts of HeLa cells transfected with BiPro-tagged mDia1 and two of its deletion fragments, containing (456-761mDia1) or lacking (1-448mDia1) the FH1 domain. HeLa cells were treated with the membrane permeant crosslinker DSP before lysis, mDia1 proteins were precipitated from the lysates with an antibody against the BiPro tag (4A6), centrifuged and subjected to SDS-PAGE and subsequent blotting. The presence of profilin I in the pellets was monitored with a monoclonal profilin antibody (2H11), which recognizes human profilin I. Note that profilin I only coprecipitates in the presence of the FH1.
