Anti mouse hrp 31430

Total protein was isolated from NSCs using M-PER extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA) and quantitated. A 30µg amount of denatured protein was resolved by SDS-PAGE and transferred to PVDF membranes, and non-specific binding was reduced by blocking in 5% non-fat milk. Primary antibodies rabbit anti-Mecp2 (1:1000, ab2829, Abcam, Cambridge, UK), rabbit anti-PSD-95 (1:1000, ab18258, Abcam, Cambridge, UK), rabbit anti-Synaptophysin (1:1000, ab32127, Abcam, Cambridge, UK), mouse anti-tau (1:1000, Tau46 #4019, Cell Signaling, Danvers, MA, USA), mouse anti-Clathrin1HC (1:1000, SC12734, Santa Cruz, TX, USA) or mouse anti-beta actin (1:5000, A1978, Sigma-Aldrich, St. Louis, MO, USA) was added to the PVDF membrane overnight at 4 °C. The next day, HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (1:1000, anti-mouse HRP-31430; anti-rabbit HRP-31460, Thermo Fisher Scientific) were added. Chemiluminescence signals were captured on X-ray films, and the bands were quantified (GS-800 densitometer, Bio-Rad, Hercules, CA, USA).

Total protein extracts were prepared using a lysis buffer (50 mM Tris, 2% SDS, 10% glycerol, 0.74 M beta-mercaptoethanol), sonicated on ice using a Sonifier 250D (Branson Ultrasonics, St. Louis MO, USA), and heated for 5 min at 99 °C. Protein concentrations were determined with the Bio-Rad DC Protein Assay Kit (BioRad, Hercules, CA, USA) using bovine serum albumin as a standard. Protein extracts were separated in 10% SDS-PAGE, electrotransferred to PVDF membranes (Immobilon-P; EMD Millipore, Burlington, MA, USA). Antibodies used are: HSP90 antibody [H90-10] #ab53497, MYC #ab32 (Abcam, Cambridge, UK) [36 (link)], #sc764 (Santa Cruz Biotechnology, Dallas, TX, USA) [37 (link)]; TUBULIN #ab6046 (Abcam, Cambridge, UK) [38 (link)]; Anti-MOUSE HRP #31430 (Thermo Fisher, Waltham, MA, USA) [39 (link)]; Anti-RABBIT HRP #31460 (Thermo Fisher, Waltham, MA, USA) [40 (link)].

VLPs were detected by ELISA with anti-AAV2 (intact particle) mouse monoclonal, A20 antibody or its single-chain derivative A20 scFv-Fc. MaxiSorp 96 well-plates (Nunc) were coated with VLPs (50 µg/ml) or rAAV2 (1.6 × 10⁹ AAV2/ml) for 1 h at 37 °C and afterwards blocked with 0.8% BSA for 1 h. Samples were incubated with A20 antibody (Progen, 1: 250 dilution), or A20 scFv-Fc (produced by our working group, 1: 250 dilution, manuscript in preparation) for another 1 h at 37 °C. Anti-mouse-HRP (31430, ThermoFisher Scientific) or anti-human-HRP (ThermoFisher Scientific) (1: 2000, 1 h incubation at 37 °C) was used for detection. Following every incubation step, the plate was washed three times with 0.05% Tween in blocking buffer. Lastly, samples were developed with 1 g/L 2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), and optical density was measured by a microplate reader (BioTek) at 405 nm.