Nupage 4 12 bis tris gels for sds page

Pre-cast NuPAGE 4–12% Bis-Tris Gels for SDS-PAGE (Invitrogen) were loaded with 20–25 μg of protein for each sample per well, run under reducing conditions, and then transferred to nitrocellulose membranes. Membranes were then blocked overnight at 4°C with 10% nonfat dried milk. The next day membranes were incubated with T22 (1:250) for tau oligomers, Tau-5 (1:1000) for total tau, and GAPDH (1:1000; Sigma) as a loading control, diluted in 5% nonfat dried milk for 1 hr at room temperature. Tau-5 and GAPDH immunoreactivity were detected with horseradish peroxidase-conjugated IgG anti-mouse secondary antibody (1:3000, GE Healthcare) and T22 was detected with horseradish peroxidase-conjugated IgG anti-rabbit secondary antibody (1:3000, GE Healthcare). For signal detection, ECL plus (GE Healthcare) was used. Densitometry of each band was quantified and normalized with GAPDH using Image-J and analyzed by one-way ANOVA.

Pre-cast NuPAGE 4–12% Bis-Tris Gels for SDS-PAGE (Invitrogen) were loaded with 10–20 μg/well of cell lysate, ran under reducing conditions, and transferred to nitrocellulose membranes. After blocking for 1 h at RT with 10% nonfat dried milk, membranes were incubated with mouse anti-EXT2 (1:1000, Santa Cruz Biotechnology; sc-514092), mouse anti-p62/SQSTM1 (1:1000, Abcam; ab56416), rabbit anti-LC3B (1:1000, Novus Biologicals; NB100-2220), rabbit anti-LAMP-2 (1:1000, Invitrogen; PA1-655), mouse anti-p-Tau (Thr231) (AT180) (1:1000, Thermo Scientific; MN1040), mouse anti-p-Tau (Ser202, Thr205) (AT8) (1:1000, Thermo Scientific; MN1020), Streptavidin-HRP (Southern Biotech; 7100-05), mouse anti-Tau-5 (1:15000, Bio Legend; 806402), rabbit anti-Tau (1:2000, Abcam; ab64193) for total tau, mouse anti-βIII-tubulin (1:1000, Abcam; ab78078) as loading control. Primary antibodies were diluted in 5% nonfat dried milk overnight at 4 °C. Immunoreactivity was detected with horseradish peroxidase-conjugated IgG anti-rabbit and anti-mouse secondary antibody, respectively (1:10,000, GE Healthcare). For signal detection, ECL plus (GE Healthcare) was used. Densitometry of each band was quantified and normalized with internal control using ImageJ (NIH).