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. 2008 Jan 14;180(1):159–172. doi: 10.1083/jcb.200707136

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Copyright © 2008, The Rockefeller University Press

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Figure 7. — STX16 interacts directly and specifically with GCC185. (A) GST-tagged cytosolic SNAREs were incubated with purified GCC185–C-110. Tagged SNAREs were recovered by glutathione–Sepharose beads, and the bound proteins were analyzed by SDS-PAGE and immunoblotting with rabbit anti-GCC185 (top) to detect bound C-110 or with rabbit anti-GST (bottom) to detect input SNAREs. Input lane at the left (top row) represents 2% of the total GCC185–C-110 used in each reaction. (B) STX16 interacts with full-length GCC185. Recombinant GST-tagged soluble SNARE proteins were incubated with K562 cell cytosol; the tagged SNAREs were then retrieved with glutathione–Sepharose beads. Bound proteins were analyzed by SDS-PAGE and immunoblotting to detect bound, full-length GCC185 (top), full-length Golgin97 (middle), or resin-bound GST-SNARE proteins (bottom). The input lane represents 1% of the total cytosol used in each reaction. (C) Quantitation of the data shown in B. Error bars represent SD from two independent experiments. (D) STX10, STX16, and Vti1a coimmunoprecipitate with GCC185. HeLa cells expressing the myc-tagged soluble SNAREs were lysed and incubated with equal amounts of either control serum or anti-GCC185 serum. Immune complexes were isolated with protein A–agarose, and bound proteins were analyzed by SDS-PAGE and immunoblotting with either rabbit anti-GCC185 serum (top) or mouse anti-myc antibody to detect exogenous SNARE proteins. A Ponceau S–stained region of the gel is shown as a loading control for IgG at the bottom. Numbers next to the gels in A, B, and D indicate M _r in kilodaltons.