Fig. 6. GATE–16 interacts with GOS–28. (A) Detergent extracts (40 μg) of rat liver Golgi membranes and His₆-GATE–16 (1 μg) were immunoprecipitated with either anti-GATE–16, anti-GOS–28 antibodies, or pre-immune IgGs as control. Immunoprecipitates were analyzed by Western blots using the indicated antibodies. Total (right panel) represents 10% of the membrane detergent extract used for each experiment in the presence of His₆-GATE–16. (B) Glutathione–agarose beads were incubated with His₆GATE–16 in the presence of recombinant GST–GOS–28 (left) or GST alone (right). The glutathione–agarose beads were washed and the bound material was eluted with free glutathione and analyzed by Western blots. (C) Detergent extracts were immunoprecipitated with anti-GATE–16 antibodies in the presence of 5 mM MgCl₂ and with either NSF (0.3 μg), αSNAP (0.8 μg), 1 mM ATP or 1 mM ATPγS, as indicated. The immunoprecipitates were analyzed by Western blots to detect co-immunoprecipitated GOS–28. Panels show immunoblots of GOS-28 (top) and GATE-16 (bottom) of the respective samples.