Fig. 8. Association of the V-ATPase membrane domain to SNARE complexes. (A) Solubilized synaptosomal proteins were incubated in parallel with different immunobeads, coated with mAbs to VAMP-2 (19K2), syntaxin-1 (HPC-1) and SNARE complexes (1567). The SNARE protein contents of immunoprecipitated (ip) and unbound (nr) samples deriving from 10 µg synaptosomal protein are compared. (B) Samples depleted of VAMP-2 (19K2 nr in A) or of syntaxin-1 (HPC-1 nr in A) were submitted to a second incubation with the immunobeads (as in A). The syntaxin-1, SNAP-25 and VAMP-2 contents of the resulting immunoprecipitated and unbound samples are compared (deriving from 20 µg synaptosomal protein). (C) Detection of the a1 and c subunits of V-ATPase in the immunoprecipitated samples deriving from the first incubation (left) or the second incubation after VAMP-2 or syntaxin-1 depletion (as indicated). Samples derive from 200 µg synaptosomal protein. (D) Parallel immunoprecipitations of VAMP-2 (19K2) or the SNARE complexes (1567) from solubilized synaptosomes (syn; 200 µg protein) or electric lobe homogenates (el; 2000 µg protein) as in (A).

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