Anti p tau ser396

dPC12 cells (5 × 10⁴ cells/per well) were treated as described above and after incubation the cells were harvested and suspended in lysis buffer (10 mM Tris, 1 mM EDTA, 1% SDS, pH 7.5). Protein concentrations were determined by the Bradford assay and equivalent amounts (10–15 μg) of total cellular proteins were separated by electrophoresis on a 12% SDS - polyacrylamide gel. Proteins were transferred to PVDV membrane and probed with anti-p-tau (Ser396; 1:800 v/v; Abcam, England) antibodies. After incubation with horseradish peroxidase–conjugated secondary antibody (1:10000; BioRad, Hercules, USA), immunoblots were developed using “Pierce ECL Western Blotting Substrate” (Thermo Scientific, USA). Membranes were stripped off and reprobed with anti-β-tubulin antibody (1:2000 v/v; Cell Signaling, USA) for loading control. Immunoblots were quantified by densitometry (ImageJ, http://rsbweb.nih.gov/ij/). Data were normalized to β-tubulin and the corresponding control was taken as 100%.

For the immunoblot assay, tau-BiFC cells grown in a 100 mm culture dish were treated with a total 12 compounds of tau-BiFC^High (Scriptaid, M344, BML281, and SAHA), tau-BiFC^Null (BML210, PhenylbutyrateNa, BML278, and Sirtinol), and tau-BiFC^Low (Aminoresveratrol sulfate, Butyrolactone 3, Salermide, and Triacetylresveratrol) groups (Tocris, Ellisville, MO, USA) at 3 µM for 36 h. Then, cell lysates were prepared by using RIPA lysis buffer containing protease/phosphatase inhibitor cocktail (Sigma), and deacetylase inhibitors (1 µM TSA and 5 mM Nicotinamide). Quantity of 10 μg of each lysate was separated on 7.5% SDS-PAGE gel and transferred to PVDF membrane. Immuno-blot analysis was performed by using Tau5 (1:2000, AbCam, Cambridge, MA, USA), anti-p-Tau(S199) (1:2000, AbCam), anti-p-Tau(Ser396) (1:2000, AbCam), anti-ac-Tau(K280) (1 µg/mL, Anaspec, Fremont, CA, USA), anti-ac-α-Tubulin(K40) (1:1000, AbCam), anti-α/β-Tubulin (1:1000, Cell signaling, Danvers, MA, USA), anti-ac-Histone H3 (1:500, Abcam), anti-Histone H3 (1:2000, Abcam), and anti-HDAC6 (1:1000, Cell Signaling) antibodies. Band intensity was quantified using Image J software (NIH). Quantification data was analyzed by Student’s t-test.