Sc 390476

Western blot analysis was performed to assess YIAD002-induced alterations of protein biomarkers in 5XFAD mice brain lysates. Twenty micrograms of lysate samples were loaded onto 12% tris-tricine gels, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were blocked by 5% skim milk or 5% BSA in TBS-T. The following primary antibodies were used for immunoblotting: anti-6E10 for APP (1:1000, Biolegend 803003), anti-AT8 (1:1000, Invitrogen MN1020), anti-Tau (1:1000, Santa Cruz SC390476), anti-ionized calcium-binding adapter molecule (Iba1) (1:1000, CST, 17198S), anti-glial fibrillar acidic protein (GFAP) (1:2000, Milipore AB5541), anti-postsynaptic density protein 95 (PSD95) (1:1000, CST 3450S), anti-synaptophysin (1:1000, Milipore MAB5258), and anti-β-Actin (1:10,000, Milipore MAB1501). Membranes were incubated with primary antibodies at 4°C overnight and probed with HRP-conjugated IgG antibodies (1:10,000) in RT for an hour. Proteins were detected using an ECL kit (Thermo Fisher Scientific, USA). Membranes were washed with TBS-T in between steps, three times for 10 min.

Cerebral cortex was lysed using RIPA buffer with a proteinase inhibitor. The samples were diluted to the same quantities (20 μg) and then loaded. Protein samples were separated by SDS-PAGE electrophoresis on 8–10% gels and then electro-transferred onto PVDF membranes (Millipore Corp). The membranes were blocked and then incubated with primary antibody (1:3,000) overnight at 4°C. Subsequently, the membrane was incubated with secondary antibodies (1:3,000 Abcam, anti-mouse IgG or anti-rabbit IgG). Then the blot was detected using the Western Bright ECL western blotting detection kit (Bio-Rad Laboratories). Equal sample loading was verified by the detection of GAPDH. The primary antibodies were as follows: mouse monoclonal anti-GAPDH (sc-66163, Santa Cruz, CA, United States), rabbit monoclonal anti-Fyn (ab125016, Abcam), rabbit monoclonal anti-SRPK2 (ab192238, Abcam), mouse monoclonal anti-tau (sc-390476, Santa Cruz, CA, United States), mouse monoclonal anti-tau (phospho Tyr18, 9G3, Genetex), and rabbit monoclonal anti- tau (phospho Ser214, ab170892, Abcam).

Samples were run on either 4–20% Express Plus PAGE in Tris-MOPS-SDS running buffer (GenScript), 10% Criterion TGX gels (Bio-Rad) in Tris-glycine-SDS running buffer, or 3–8% Criterion XT Tris acetate gels (Bio-Rad) in Tris acetate-SDS running buffer. Proteins were transferred to PVDF membranes (Trans-Blot Turbo RTA transfer kit, Bio-Rad) using the Trans-Blot Turbo transfer system (Bio-Rad) and immunoblotted with the Tau antibody A-10 (1:1000–10,000, mouse, sc-390476, Santa Cruz Biotechnology) or anti-GFP (1:10,000, mouse, MMS-118P, Covance). An alkaline phosphatase-coupled secondary antibody (Vector Laboratories) together with ECF substrate (GE Healthcare Life Sciences) was used for development. The blots were imaged on an ImageQuant LAS-4000 (FUJIFILM Co.). Densitometric quantification of the signals was performed with Image Studio Lite software (LI-COR Biosciences).