Fig. 7. Complex formation between syndapin II and dynamin II is required for vesicle formation from isolated Golgi membranes. (A) Vesicle formation from Golgi-enriched membranes of HepG2 cells is inhibited in a dose-dependent manner by the addition of the GST-tagged SH3 domain of the dynamin II-binding proteins syndapin II (circles) and amphiphysin II (squares). The assay contained Golgi-enriched membranes, ATP, GTP and 40 µg cytosolic proteins as described in the Materials and Methods. Vesicles formed were sedimented and the radioactivity of newly synthesized proteins that were packed into the vesicles was determined. The value obtained under standard conditions is arbitrarily set to 100. The experiments shown were performed in triplicate and s.d. are given. (B) The anti-syndapin II antiserum 3685 inhibits vesicle formation at Golgi-enriched membranes in a dose-dependent manner (open circles). When syndapin-specific antibodies were depleted from the antiserum 3685, inhibition was markedly reduced (filled squares). (C) Specificities and affinities of the original (left) and the depleted antiserum 3685 (right) are shown by immunostaining of western blots with 75 ng (lanes 1,4) and 15 ng (lanes 2,5) MBP-syndapin II antigen [MBP-Sdp II-I (305-388)] as well as with 30 ng (lanes 3,6) MBP-full-length syndapin I (MBP-Sdp I).