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Co-immunoprecipitation of SV2A and B by selected HCA mutants was performed by incubating 12 μg of 3xFlag-tagged wild-type and mutant H6F3HCAS, H6F3HCES, H6F3HCFS and H6F3HCGS with 6 μg anti-Flag-tag M2 antibody and Triton X-100 solubilised rat brain synaptosomal membranes. Bound SV2A and B were detected by western blotting, amount of precipitated H6F3HCXS was verified by HRP conjugated StrepMAB classic. MWM denotes molecular weight marker.
