Anti tau5

Brain samples were homogenized in ice‐cold RIPA lysis buffer. Protein samples were loaded on a 4%–20% SDS‐polyacrylamide gel and then transferred onto nitrocellulose (NC) membranes. After blocking with 5% fat‐free milk, the NC membranes were incubated with the following primary antibodies overnight at 4°C: anti‐APP C‐terminal (1:1000, Biolegend); anti‐6E10 (1:400, Biolegend); anti‐RAGE (1:1000, Millipore); anti‐LRP‐1 (1:1000, Abcam); anti‐pS396‐tau (1:1000, Abcam); anti‐pT231‐tau (1:1000, Signalway); anti‐Tau5 (1:1000, Millipore); anti‐Synapsin‐1 (1:1000, Abcam); anti‐PSD95 (1:1000, Millipore); and anti‐β‐actin (1:2000, Origene). The membranes were incubated with the corresponding IRDye 800 CW‐conjugated secondary antibodies and scanned using an Odyssey fluorescent scanner. Relative band intensities were normalized to the band intensity of the internal reference protein for analysis.

Protein concentrations were quantified using the Bradford Assay (Bio-Rad Protein Assay 500-0006, Munchen, Germany) 5 μg of TIF extracted proteins were separated by 10% SDS polyacrylamide gel electrophoresis. PVDF membranes were blocked in Tris-buffered saline (5% non fat milk powder, 0.1% Tween20, 1 h, room temperature). Primary antibodies were diluted in the same buffer (incubation overnight, 4 °C) using: APP (cat. #2024170, 1:1000, Millipore, Billerica, MA, USA), p-APP (cat. #MABN10, 1:1000, Millipore, Billerica, MA, USA), anti Tau-5 (cat. #MABN162, 1:1000, Millipore Mab 361), p-Tau (cat. #MABN388, 1:1000, Millipore, Billerica, MA, USA), c-Jun (cat. #9165, 1:1000, Cell Signaling Technology, Danvers, MA, USA), p-c-Jun [Ser63] (cat. #9164, 1:1000, Cell Signaling Technology, Danvers, MA, USA),p-JNK (cat. #9251, 1 : 1000, Cell Signaling Technology, Danvers, MA, USA), JNK (cat. #9252, 1 : 1000, Cell Signaling Technology), anti-Actin (cat. #MAB1501, 1:5000, Millipore, Billerica, MA, USA) and at least six independent experiments were performed. Blots were developed using horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and the ECL chemiluminescence system (Promega). Western blots were quantified by densitometry using ImageQuant TL software (Amersham Biosciences, Amersham, UK) and was based on at least three independent experiments.