Goat anti rabbit or anti mouse secondary antibodies conjugated to horseradish peroxidase

Equal proportions of each sample were loaded on 10% polyacrylamide gels and resolved by gel electrophoresis. After electrophoretic transfer to nitrocellulose membranes, the blots were blocked in 5% milk with Tris-buffered saline for an hour. For phosphorylation-specific antibodies, the blots were blocked in 5% bovine serum albumin (BSA). Primary antibody was added and incubated overnight at 4°C at 1: 1000 dilution for 3026 tau antibody (Strang et al., 2017 (link); Xia et al., 2021a (link)) or β-tubulin antibody (clone TUB 2.1, Fisher Scientific, Waltham, MA). Phosphorylation-specific tau antibodies 7F2 and AT180 were used to detect tau phosphorylated at Thr205 and Thr231, respectively (Goedert et al., 1994 (link); Strang et al., 2017 (link)). The next day, the samples were incubated in goat anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase (Jackson Immuno Research labs, Westgrove, PA) for an hour. After TBS washes, the membranes were exposed and imaged using Western Lightning Plus ECL reagents (PerkinElmer, Waltham, MA). Each immunoblot image was imported into ImageJ program (National Institutes of Health, Bethesda, MD) for densitometric analysis. Statistical analysis was performed with Graphpad Prism software (San Diego, CA) and one way ANOVA with Dunnett’s test was used to calculate group comparisons.

Equal proportions of each sample were loaded on 10% polyacrylamide gels and separated by SDS-PAGE. After transfer, the membranes were blocked in 5% milk with Tris-buffered saline (TBS) for an hour. The membranes were incubated in primary antibody overnight at 4 ºC at dilutions of 1:1000 for β-tubulin and tau antibodies. Anti-β-tubulin (Clone TUB 2.1) is a mouse monoclonal antibody (Sigma-Aldrich, St. Louis, MO) and 3026 tau antibody is a rabbit polyclonal antibody that was raised against full length 0N3R human tau but also reacts with 0N4R human tau (Supplemental Fig. 1)^{26 (link),42 (link),43 (link)}. After TBS washes, goat anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase (Jackson Immuno Research labs, Westgrove, PA) were added to the membranes at 1:4000 dilution for an hour. After several washes, the membranes were exposed and imaged after adding Western Lightning Plus ECL reagents (PerkinElmer, Waltham, MA). Each lane was semi-quantitatively measured in ImageJ by densitometric analysis. Statistical tests were performed on GraphPad Prism version 8.4.3 for one way analysis of variance (ANOVA) with post hoc analysis and Dunnett’s test.