Briefly, AAV or T4-AAV particles were boiled in loading buffer for 10 min, separated by 12% SDS-PAGE, and then transferred to nitrocellulose membranes (Bio-Rad, CA). Blocking was performed in 5% bovine serum albumin (BSA)/PBS-T buffer [PBS (pH 7.4) and 0.05% Tween 20] at RT for 1 hour with gentle shaking. Blots were then washed three times with PBS-T. Primary antibodies were added to the blots and incubated overnight at 4°C in PBS with 5% BSA. AAV VP proteins were detected using the AAV capsid protein–specific antibody B1 (diluted 1:50; American Research Products, MA), which recognizes VP1, VP2, and VP3 on the basis of their identical C-terminal regions. After washing with PBS-T three times, a secondary goat anti-mouse HRP-conjugated antibody (Thermo Fisher Scientific, MA) was applied at a 1:2000 dilution in 5% BSA/PBS-T for 1 hour at RT, followed by rinsing three times with PBS-T. The bioAAV, bioSoc, and Hoc-BAP-biotin were directly detected by HRP-conjugated streptavidin (Abcam, UK). Signals were visualized with an enhanced chemiluminescence substrate (Bio-Rad, CA) using the Bio-Rad Gel Doc XR+ System and Image Lab software according to the manufacturer’s instructions (Bio-Rad, CA).

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