Virus Overlay Assay for Capsid Binding

The virus overlay assay was performed as previously reported [34 (link)] with some modifications. Briefly, protein lysates were separated on Bolt 4–12% Bis-Tris Plus gels and transferred onto nitrocellulose membranes. After incubation with AAV9 or PHP.eB at 5e11 vg/mL, the membranes were fixed with 4% PFA at room temperature for 20 minutes to crosslink the interaction between the capsid and its target protein, followed by 2M HCl treatment at 37°C for 7 minutes to expose the internal capsid epitope for detection. The blots were then rinsed and incubated with anti-AAV VP1/VP2/VP3 (1:20; American research products, Inc., cat. #03–65158), anti-LY6A (1:1000; BD, 553333 or 557403) or anti-alpha-tubulin (1:1000; Sigma, T9026) followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody at 1:5000. The detection of the HRP signal was by SuperSignal West Femto Maximum Sensitivity Substrate using a Bio-Rad ChemiDoc ^TM MP system #1708280.