Western blotting and co-immunoprecipitation

Cultured cells or hippocampus tissues were collected on ice, and homogenized with RIPA lysis buffer containing 50 mM Tris-HCl, 100 mM NaCl, 1% Triton X-100, 5 mM EDTA, and 1:100 PMSF, and then centrifuged at 12,000 × g for 20 min. The supernatant was collected for the analysis of soluble proteins. In some experiments, the pellets were further incubated with 8% SDS buffer at 37 °C and then ultrasound for 60 times to reach a complete resuspension for the analysis of insoluble tau. Protein concentration was measured through BCA assays (Thermo Fisher). Equal amount of protein from each sample were separated in SDS-PAGE gels, and then transferred onto nitrocellulose or 0.22 μm polyvinylidene fluoride membranes (Merck Millipore). The membranes were blocked with 5% BSA, and incubated in turn with primary and horseradish peroxidase-conjugated secondary antibodies: pS199-Tau (44734G, Invitrogen, 1:2000), pT205-Tau (11108-1, Signalway, 1:2000), pS396-Tau (11102, Signalway, 1:2000), pS404-Tau (11112, Signalway, 1:2000), AT8 (MN1020, Thermo Fisher, 1:1000), Tau5 (ab80579, Abcam, 1:1000), Tau46 (T9450, Sigma, 1:1000), PP2A-Bα (4953, Cell signaling, 1:1000), Phospho-Ser/Thr (ab17464, Abcam1:1000), Ubiquitin (ab7254, Abcam, 1:1000), MAP2 (AB5622, Millipore, 1:2000), hTau-N368 (a generous gift from Prof. Keqiang Ye, Emory University School of Medicine, 1:1000), DEPTAC (developed by Daian Biotechnology, Wuhan, China, 1:500), β-actin (ab6276, Abcam, 1:2000). HRP-conjugated goat anti-rabbit IgG (A0208, Beyotime, 1:3000), HRP-conjugated goat anti-mouse IgG (A0216, Beyotime, 1:3000). Blots were visualized using ECL luminol reagent (P0018FS, Beyotime), and quantified using ImageJ software.

For co-immunoprecipitation, cultured cells were lysed on ice with RIPA buffer for 10 min containing 1:100 PMSF. Protein samples were incubated with protein G agarose (Beyotime) at 4 °C for 2 h, and then centrifuged for 5 min (4 °C, 12,000 rpm). Supernatants were subsequently incubated with specified antibodies and protein G agarose overnight at 4 °C. The agarose beads were washed three times with PBS, and resuspended in 30 μL buffer containing 2% SDS, 100 mM dithiothreitol, 10% glycerol, and 0.25% bromophenol blue, and then denatured at 95 °C for 5 min. Immunoprecipitants were subsequently analyzed through Western blotting.