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. Author manuscript; available in PMC: 2013 Jan 19.
Published in final edited form as: Cell Host Microbe. 2012 Jan 19;11(1):46–57. doi: 10.1016/j.chom.2011.11.009

Figure 5. DrrA coordinates the functional pairing of Stx3 and Sec22b in permeabilized cells.

Figure 5

Permeabilized HEK293-FcγRII cells producing the indicated GFP-Stx3 and GFP-DrrA proteins were incubated with vesicles displaying 3x-FLAG-Sec22b and protein complexes were precipitated using anti-FLAG agarose. (A) The GFP-DrrA and 3x-FLAG-Stx3 constructs expressed in the permeabilized HEK293-FcγRII cells and the inclusion of GTP or GDP in the reactions are indicated above each lane. Immunoblot analysis using antibodies specific for GFP, SNAP23, and FLAG show protein levels in the reaction (5% or input, left panels) and protein levels in the anti-FLAG immunoprecipitate (IP, right panels). Because SNAP23 has been shown to form a SNARE complex with Sec22b and Sec18, this interaction served as a positive control. (B) As in panel A, HEK293-FcγRII cells producing GFP-Stx3 and GFP-DrrA61-647 were permeabilized and incubated with vesicles in a PNS fraction obtained from cells producing 3x-FLAG-Sec22b in the presence of GTP. Lysates of these reactions were incubated in the presence or absence of purified NSF and α-SNAP as indicated. Protein levels in the reaction (5% or input) and protein levels in the 3x-FLAG-Sec22b immunoprecipitate (IP) were determined by immunoblot analysis using antibodies specific for GFP, SNAP23, and FLAG.