Mn1020

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.

Phosphatase inhibitor cocktail IV (ab201115) and antibody against GAPDH (ab181602) were purchased from Abcam. Primary antibodies against phosphorylated tau at Ser396 (PHF13; sc-32275), Ser202/Thr205 (AT8; MN1020) and Thr212/Ser214 (AT100; MN1060) were procured from Santa Cruz Biotechnology and Thermo Scientific, respectively. Secondary antibodies IRDye^® 800 CW goat anti-mouse (926-32210) and IRDye^® 680 RD goat anti-rabbit (926-68071) were sourced from LI-COR Biosciences. Immunohistochemical biotinylated link and streptavidin/HRP complex solutions (LSAB2 System-HRP Kit, K0675) were from Dako. Protease inhibitors pepstatin A (P5318), leupeptin hydrochloride (L9783) and phenylmethanesulfonylfluoride (PMSF; P7626), 3,3’-diaminobenzidine and H₂O₂/urea tablets (D4293-50SET) and DPX mounting media (44581) were from Sigma–Aldrich. Other reagents used in this study were of analytical grade and procured from either Thermo Fisher Scientific or Sigma–Aldrich.