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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 4. DrrA and Rab1 coordinate Stx3 interactions with Sec22b.

Figure 4

The binding of purified MBP-Stx3 ΔHab/TMD to vesicle-associated 3x-FLAG-Sec22b was assayed using amylose agarose to pull down MBP protein complexes. (A) Purified MBP-Stx3 ΔHab/TMD was incubated in vitro with the purified His-DrrA derivatives, GST-Rab1 and nucleotides indicated in the grid above each lane. A PNS fraction containing vesicles displaying 3x-FLAG-Sec22b was incubated with each reaction. Proteins in the reaction (15% or 5% of input) and in the MBP pull-down were separated by SDS-PAGE and detected using antibodies specific for either the His, GST, FLAG or MBP tag as indicated on the right of each blot. (B) Blots from the experiment shown in panel A were scanned to determine relative amounts of 3xFLAG-Sec22b associated with MBP-Stx3 ΔHab/TMD. Relative amounts of 3x-FLAG-Sec22b were calculated by dividing the signal intensity observed for reaction conditions indicated below each bar by the signal intensity in the control lane where MBP-Stx3 ΔHab/TMD alone was used to pull-down 3x-FLAG-Sec22b on vesicles. Data represent the mean ± SEM of four independent experiments.