P tau thr181

Cells were lysed in RIPA buffer with 1% phenylmethylsulfonyl fluoride and 1% protein phosphatase inhibitor (Beyotime, China) on ice for 20 min. Proteins in the mouse tissue homogenate were extracted with RIPA buffer (Beyotime, China). Lysates were centrifuged at 12,000 rpm for 30 min at 4°C to obtain supernatants. Equal amounts of protein lysates in RIPA were separated on 8%, 10% or 12% SDS-PAGE gels before being transferred to polyvinylidene fluoride membranes (Merck Millipore, USA). The membranes were blocked with 5% milk and then incubated with primary antibodies in primary antibody dilution buffer (Beyotime, China) at 4°C overnight. The blots were probed with the following antibodies: Tau 1:1,000, P-Tau Thr181 1:1,000, Akt 1:1,000, P-Akt Ser 473 1:2,000, P-ERK1/2 Thr202/Tyr204 1:2,000, ERK1/2 1:1,000, Caspase 3 1:1,000, P-PKCζ/λ Thr410/403 1:1,000 (Cell Signal Technology, USA); KIBRA 1:500, PKCζ 1:1,000 (Santa Cruz, USA); PARP-1 1:1,000 (Abcam, UK); β-actin 1:2,000 (Proteintech, China). The membranes were subsequently incubated with peroxidase-conjugated secondary antibodies and developed using the enhanced chemiluminescence (Merck Millipore) method. Band densities were quantified by densitometry using Fluor ChemQ software.

The OCT-embedded brains were cryosectioned at a 30 μm thickness. Staining was performed according to the previously reported method with a slight modification [50 (link),51 (link)]. The sections were permeabilized and blocked with 5% normal goat serum in 1X Tris-buffered saline (TBS) + 0.1% Triton-X (#9002-93-1, Millopore Sigma, St. Louis, MO, USA) for 1 h at room temperature. The sections were then incubated with a primary antibody against pTau Thr181 (1:500, #12885, Cell Signaling Technologies, Danvers, MA, USA) overnight at 4 °C and washed thoroughly with 1X TBS 3 times for 5 min. The sections were incubated in anti-rabbit AlexaFluor 488 (1:1000, #A11034, Invitrogen, Waltham, MA, USA) secondary antibody for 1 h at room temperature before washing with the same procedure. The sections were then stained with AlexaFluor 647 conjugated anti-NeuN (1:500, #D4G40, Cell Signaling Technologies, Danvers, MA, USA) antibody overnight at 4 °C before washing and treating with Hoechst (1:10,000, #33342, Invitrogen, Waltham, MA, USA). The sections were washed for 5 min 3 times with 1X TBS, coverslipped with Prolong Glass Antifade mountant (#P36930, Invitrogen, Waltham, MA, USA) and dried overnight at room temperature before storing at 4 °C. The slides were imaged on an LSM 800 confocal microscope (Zeiss, Jena, Germany).

Antibodies to human CD45 (CD45-PE-Cy7, catalog [cat] #304016), CD8 (CD8-APC, cat #344722), and CD3 (CD3-Pacific blue, cat #300330) were purchased from Biolegend (San Diego, CA). Antibodies to human CD4 (CD4-FITC, cat #555346) were from BD Biosciences (San Jose, CA); human leukocyte antigen (HLA)-DR (cat #NB100-77855SS), and Aβ1–42 (cat #NBP2-13075SS) from Novus Biologicals (Centennial, CO). Antibodies for microtubule-associated protein-2 (MAP 2, cat #ab32454), neurofilament-L (NFL, cat #ab223343), NeuN (cat #ab177487), claudin-5 (cat #ab15106), zonula occludens (ZO)-1 (cat #61–7300), β-actin (cat #ab8226), LRP1, (cat #ab92544), RAGE (cat #ab216329), phospho(p)-Tau (Ser396) (cat #ab32057), pTau (Ser199) (cat #ab4749), pTau (Thr231) (cat #ab151559), and pTau (Thr205) (cat #ab254410) were from Abcam (Cambridge, MA). Antibodies for ZO-2 (cat #71–1400) were from Invitrogen (Carlsbad, CA); Tau (cat #46687S) and p-Tau (Thr181) (cat #12885S) from Cell signaling Technologies (Danvers, MA).