Protein samples were separated on SDS-polyacrylamide mini-gels and blotted onto PVDF membranes (Millipore). As primary antibodies, rabbit polyclonal anti-Stx7 (Bethyl Laboratories), mouse monoclonal anti-Syb2 (Cl69.1), and mouse monoclonal anti-Tuj1 (Covance) were used. Blots were further incubated with secondary antibodies (anti-mouse IgG or anti-rabbit IgG, respectively) coupled to horseradish-peroxidase and developed using a Western Lightning Plus-ECL kit (PerkinElmer). Signals were detected using a Molecular Imager ChemiDoc (BioRad).
To estimate the copy number of Stx7 per SV, the CPG-purified SV fraction (a gift from Reinhard Jahn) with known protein concentration and vesicle number, was subjected to western blotting together with various amounts of standard proteins for Stx7 and Syb2. Band intensities in acquired images were quantified using Quantity One software (BioRad).
To estimate the copy number of Stx7 per SV, the CPG-purified SV fraction (a gift from Reinhard Jahn) with known protein concentration and vesicle number, was subjected to western blotting together with various amounts of standard proteins for Stx7 and Syb2. Band intensities in acquired images were quantified using Quantity One software (BioRad).