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Figure 4


Fig. 4. Apical SNARE complex in MCD4 cells. Anti-Stx3 antibodies or normal rabbit IgG were used for co-immunoprecipitation studies from MCD4 cell lysates. (A) Immunocomplexes were resolved by NuPAGE and immunoblotted with antibodies against Stx3, VAMP2, VAMP3, SNAP23, Munc18b and Munc18c. Anti-Stx3 antibody was very efficient in immunoprecipitating Stx3 protein from MCD4 lysates and no bands were seen in samples incubated with normal rabbit serum (Rb IgG). Immunoblotting performed on the same samples revealed that equal amounts of VAMP2 and VAMP3 co-precipitated with Stx3 in both resting or FK-stimulated MCD4 cells, whereas the association of SNAP 23 or Munc18b with Stx3 was FK-dependent. No band for Munc18c was detectable in Stx3 immunoprecipitates, even after long exposure. (B) Densitometric analysis of the amount of each protein precipitated from contol- (ctr; set as 100%) or FK-stimulated cells. Experiments were performed three times with comparable results.





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