Western Blot

All cells analyzed using western blot were incubated with non-tagged 6B2 antibodies and/or PHF. All samples were homogenized in RIPA buffer and prepared as described previously [14]. Samples were boiled and loaded on 10% SDS-PAGE gels, electrophoresed, and then transferred to nitrocellulose membranes, which were blocked in 5% milk with 0.1% TBS-T. Blots were then probed for total tau (Dako polyclonal antibody), phospho-tau (PHF-1 monoclonal antibody) or GAPDH (Abcam polyclonal antibody) primary antibodies overnight at 4°C, washed and then probed with anti-horseradish peroxidase (HRP) conjugated rabbit or mouse secondary antibody (Pierce) for 1 h. For antibody uptake detection, membranes were incubated with an anti-mouse IgG1 HRP-conjugated secondary antibody with specificity against the heavy chain (Bethyl Laboratories), and signal was detected with an ECL substrate (Thermo Scientific). Images of immunoreactive bands were then acquired and quantified using the Fuji LAS-4000 imaging system.