Figure 3.

Dock180 directly binds to GRASP. A) Localization of the GRASP-binding domain in Dock180. Hek 293 cells expressing HA-GRASP and the indicated VN-flag-Dock180 constructs were lysed, and incubated with M2 anti-flag resin as described in Materials and Methods. The immunoprecipitates as well as saved samples of the starting lysate were blotted with goat anti-Dock180 and mouse anti-HA. B) Localization of the Dock180-binding domain in GRASP. Hek 293 cells expressing HA-GRASP or a version lacking the ala/pro rich region (del82-GRASP) and flag-Dock180 constructs were lysed, and incubated with M2 anti-flag resin as described in Experimental Procedures. The immunoprecipitates as well as saved samples of the starting lysate were blotted with goat anti-Dock180 and mouse anti-HA. C) GRASP directly binds the SH3 domain of Dock180. E. coli were transformed with pGEX2T-GRASP or pST50Tr-FLAGyGcn5x-Flag-Dock180 (11KDa). The expressed proteins were purified as described in Materials and Methods. GST-GRASP (70 KDa) was eluted from the Sepharose resin using a solution of Glutathione. It was then added to either M2-Flag Sepharose gel or M2-Flag-Dock180 beads. The resin was washed three times and attached proteins were eluted in 30μl sample buffer. GRASP and Dock180 were detected by GelCode (Coomassie) staining and Western blotting with Goat anti-GRASP as well as goat anti-Dock180 antibodies (N-terminal).

Attar and Santy BMC Cell Biology 2013 14:9   doi:10.1186/1471-2121-14-9
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