Anti pak

Tissues from 3xTg‐AD mice and AD patients were homogenized with a douncer and sonicated in 200 μl of RIPA with a protease inhibitor cocktail (Complete, Mini EDTA‐free tablets, Roche, Mannheim, Germany). Astrocyte (1×10⁵) and tissue homogenates were centrifuged at 4°C for 10 min at 12,000 × g, and protein content in the supernatant was quantified with the Bio‐Rad Protein Assay (Bio‐Rad, Hercules, CA, USA). Cell and tissue extracts (10 μg) were loaded into gels and blots were developed with rabbit polyclonal anti‐integrin β1, antiphosphoPAK, anti‐PAK, antiphosphoPKC, antiphosphoPKD, antiphosphoAKT, anti‐AKT (1:1000, Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal anti‐NOX2, anti‐NOX‐1 (1:1000, Novus Biologicals, Littleton, CO, USA), rabbit polyclonal anti‐β actin (1:5000, Sigma, St. Louis, MO, USA), mouse monoclonal anti‐GFAP, anti‐Rac1, anti‐GAPDH (1:2000, Millipore Ibérica, Madrid, Spain), and anti‐beta amyloid 1–16 (1:1000, 6E10 BioLegend, San Diego, CA, USA). For dot blot assay, human tissue extracts (2 μg) were spotted into a nitrocellulose membrane, and rabbit polyclonal anti‐Aβ1‐42 (1:10000, Abcam, Cambridge, UK) was used for chemiluminescence detection.

Protein from cell or tissue lysates was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane (Pall Corp, Port Washington, NY). Then the membranes were blocked with 5% skimmed milk and incubated using primary antibodies anti-CSRP2 (1:800 dilution, Sigma), anti-GAPDH, anti-p130Cas, anti-p-p130Cas (Tyr410), anti-PAK, anti-p-PAK, anti-LIMK, anti-p-LIMK, anti-cortactin, anti-p-cortactin, anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-β-catenin (Cell Signaling Technology, Beverly, MA), anti-LATS1, anti-Yap (Proteintech), anti-ERK1/2, anti-p-ERK1/2 (Thr202/Tyr204), anti-p-Yap (Ser127) (Absci), anti-p-LATS1 (Thr1079/1041) (Bioss) at 4 °C. Then the membranes were incubated with appropriate secondary antibodies (anti-rabbit IgG, 1:3000 dilution, #7074; cell signaling). Enhanced chemiluminescence (Pierce, Rockford, 1L, USA) was used to detect the signal.