Binding of γ-adaptin and ARF1 by Syt IX. (A) Twenty micrograms of GST, GST-Syt IX-C2A, GST-Syt IX C2B-CT, or GST-Syt IX C2BΔCT was immobilized on glutathione-Sepharose beads and incubated for 18 hours at 4°C with RBL cell lysates (500 μg). Bound proteins were eluted in sample buffer; half of the total eluted proteins were resolved by SDS-PAGE and analyzed by Western blot using anti–γ-adaptin antibodies. Total lysate (TL; 100 μg) was loaded. (B) Twenty micrograms of GST, GST-Syt IX-C2A, GST-Syt IX-C2B-CT, or GST-Syt IX C2BΔCT, as indicated, was immobilized and incubated as described except that RBL cells lysates (500 μg) were derived from cells transiently transfected with ARF1-GFP cDNA. Bound proteins were eluted in sample buffer; half of the total eluted proteins were resolved by SDS-PAGE and analyzed by Western blot using anti-GFP antibodies. Total lysate (TL; 100 μg) was loaded. (C) Immunoprecipitation was performed as described in “Materials and methods,” using RBL cell lysates derived from cells cotransfected with T7-Syt IX and ARF1-GFP or only ARF1-GFP cDNAs as indicated and using anti-T7 antibodies (IP antibody). Immune complexes were separated by SDS-PAGE and analyzed by Western blot using anti-GFP or anti–Syt IX antibodies as indicated (IB antibody). (D) Twenty micrograms of GST, GST-Syt IX-C2A, or GST-Syt IX-C2B-CT, as indicated, was immobilized on glutathione-Sepharose beads and incubated for 18 hours at 4°C with buffer (lanes 1 and 6), RBL cell lysates (500 μg, lane 2) DEAE Sephadex A25 effluent derived from ARF1-transformed bacteria (500 μg; lanes 3, 5, and 7) or nontransformed bacteria (500 μg; lane 4). Bound proteins were processed as described.