Mouse anti tau5

We used the following primary antibodies for this study: mouse anti-4G8 (1:1,000, BioLegend, San Diego, CA, USA), mouse anti-6E10 (1:1,000, BioLegend, San Diego, CA, USA), rabbit anti-APP N-terminus (1:1,000, Sigma, Ronkonkoma, NY, USA), rabbit anti-ADAM8 (1:200, LSBio, Seattle, WA, USA), rabbit anti-ADAM9 (1:200, Abcam, UK), rabbit anti-ADAM10 (1:200, Abcam, UK), rabbit anti-ADAM12 (1:200, Abcam, UK), rabbit anti-ADAM17 (1:200, Abcam, UK), mouse anti-BACE1 (1:200, Abcam, UK), rabbit anti-neprilysin (1:200, Millipore, Burlington, MA, USA), mouse anti-AT100 (1:500, Invitrogen, Carlsbad, CA, USA), mouse anti-AT180 (1:500, Invitrogen, Carlsbad, CA, USA), and mouse anti-Tau5 (1:500, Invitrogen, Carlsbad, CA, USA).

The hippocampus and cortex of mice were extracted and lysed by RIPA buffer (Beyotime, P0013B, China) containing 1X halt™ protease and phosphatase inhibitor single-use cocktail (Invitrogen, 78,443). Afterward, Pierce™ BCA Protein Assay Kit (Invitrogen, 23,225) was used for quantitation. Samples were separated on SDS-PAGE gel and transferred to PVDF membranes (Millipore). Then we blocked the membranes with 5% milk for one hour at room temperature and incubated with primary antibodies. The primary antibodies we used include mouse-anti-β-Tubulin (1:5000, Proteintech, 66,240–1-Ig), rabbit-anti-PDGFRβ (1:1000, Abcam, ab32570), rabbit-anti-ZO1 (1:1000, ThermoFisher, 61–7300), rabbit-anti-Occludin (1:1000, ThermoFisher, 71–1500), rabbit-anti-Claudin-5 (1:1000, Abcam, ab131259), mouse-anti-tau5(1:5000, Invitrogen, AHB0042), and Phospho-Tau Family Antibody Sampler Kit (CST, 96628 T). Specific secondary HRP-linked antibodies (1:2000, Cell Signaling Technology, 7074S or 7076S) were subsequently incubated for an hour at room temperature.