Fig. 2. Syndapin II interacts with different splice variants of dynamin II through direct association between the SH3 domain and the PRD. (A) Immunoblot analyses of co-precipitations of GFP-tagged full-length and truncated dynamin (Dyn) II proteins overexpressed in HEK293 cells with immobilized GST (–) or a GST fusion protein of the SH3 domain of syndapin II (+). Lysates as well as co-precipitated proteins were analyzed by immunoblotting with anti-GFP antibodies. The three GFP-tagged dynamin II splice variants were all specifically co-precipitated by the syndapin II SH3 domain, whereas dynamin II lacking the PRD or GFP alone did not bind (right panel). (B) A S-peptide-tagged fusion protein containing the dynamin II PRD specifically binds endogenous syndapin II from HepG2 cell extracts, whereas a related fusion protein comprising the dynamin II PH domain does not. Eluates were analyzed by immunoblotting with anti-syndapin II antibody 2521. (C) In vitro reconstitution of the interaction with purified fusion proteins (GST-Syndapin II SH3 and S-Dynamin II PRD) demonstrates that the association of both proteins is direct. SDS-eluted proteins were analyzed by immunoblotting using the anti-dynamin II antibody C12 and anti-GST antibodies to detect the S-PRD and the GST-SH3 fusion proteins, respectively.