Figure 6.

Amph1 and Amph2 form heterodimers in vivo. (A) Immunoprecipitation of Amph1 and Amph2 from brain as an equimolar complex. Rat brain extract in buffer A was immunoprecipitated with antiserum to either Amph2 or, as a control, dynamin. Bound proteins were analyzed by Coomassie blue staining. Note the presence of dynamin in both immunoprecipitates (see Figure 8A). (B) Immunoprecipitation of the two isoforms as a complex in COS cells. COS cells were transiently transfected with Amph1, Amph2, or the two together. Extracts of each were separately immunoprecipitated with antibodies against Amph1 or Amph2 and processed for Western blotting. Total proteins were also loaded to show the starting material. (Due to the strength of the Amph2 antibody, total brain (lane 1) appears to have a much higher level of Amph2 compared with Amph1, but as we have shown in Figure 2A, they are present in a 1:1 ratio.) (C) Cross-linking with DTSSP suggest the Amph1–Amph2 oligomer is a heterodimer. Rat brain cytosol (10 mg/ml) was incubated with different concentrations of DTSSP and separated by SDS-PAGE on 7% gels before blotting with the antibodies indicated.