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Fig. 9. Sbr I complexes with SNAP25 and syntaxin 1 in TGNs. The pelleted cells were extracted in 1 ml of buffer containing 1% (v/v) Triton X-100 (see Materials and Methods) for 1 hour at 4°C, followed by centrifugation. The supernatant was incubated at 4°C overnight with anti-Sbr I IgG coupled to protein A agarose. After sedimentation and extensive washing with the extraction buffer, beads were suspended in SDS sample buffer for SDS-PAGE (with and without boiling the samples for 10 minutes) under non-reducing conditions, followed by western blotting using antibodies specific for each SNARE. Only the lower halves of the gels blotted for Sbr I or Sbr II are shown because of excessive staining of the rabbit IgG that overlapped the SNARE complex. Note that boiling raised the proportions of dissociated SNAP25, syntaxin I and Sbr I; this corresponds to the decrease in the complex.
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