Sc 73614

Rat atrium tissue lysate and whole cells were prepared by 1x cell lysis (Cell Signaling Technologies, USA) and 1x protease inhibitors (Cat. 04693159001; Roche Molecular Biochemicals, USA). The crude extracts were centrifuged at 13000×g, 4 °C for 10 min and the supernatant was collected. The insoluble sediment was resuspended in 8 mM guanidine hydrochloride in complete lysis. The protein concentration was measured by bicinchoninic acid protein assay and equal amounts of protein samples were separated on 12% polyacrylamide gels, transferred onto PVDF membrane. Membranes were blocked in 5% non-fat milk or 3% BSA at room temperature and incubated with primary antibodies overnight at 4 °C. The primary antibodies targeted against Caspase-3 (Cell Signaling Technology, 9662s), BCL-2 (Abcam, ab32124), BAX (Abcam, ab32503), Vinculin (Santa Cruz Biotechnology, sc-73614), LAMP-2A (Abcam, ab125068), LC3B (Abcam, ab192890), p62 (Abcam, ab109012), MYL4 (Abnova, HOOOO4635-M01), Cathepsin B (Abcam, ab214428) or Cathepsin L (Santa Cruz Biotechnology, sc-390367) at a 1:1000 dilution. After 1-h HRP-conjugated secondary antibodies incubation, bends were visualized using chemiluminescence (ECL, TANON, China) and viewed under Amersham Imager 600 system (GE Healthcare, USA). The relative intensity of each band was normalized to Vinculin.

Total protein lysates were prepared using NE-PER Kit (Pierce, Rockford, IL, USA) and supplemented with 1X of protease inhibitor cocktail (Roche) according to the manufacturer’s instruction. For detection of proteins 25 μg of total extracts were mixed with denaturing buffer (1× Laemmli loading buffer with 10% of β-mercaptoethanol) and analyzed by SDS–PAGE/Western blot. Separated proteins were transferred onto a 0.45-μm pore size nitrocellulose membrane (Millipore) using the iBlot transfer system (Novex, Thermo Fisher, USA). Membranes were incubated with primary antibodies diluted from 1:500 to 1:1000, followed by TBS-T washes and incubation with HRP (horseradish peroxidase)−conjugated secondary antibodies (GE Healthcare). The signal was visualized by enhanced chemiluminescence with Luminata Forte Western HRP Substrate (Millipore) and the ImageQuant LAS 4000 imaging system. The following antibodies were used: anti-p-TauS262 (Abcam, ab131354), anti-total Tau (Abcam, ab76128), anti-p-Tau-T205 (Santa Cruz biotechnology, sc-73614), anti-VCL (Abcam, ab254410) and anti-p-Tau Y18 (GeneTex, GTX54658), Anti-CFL-1 (Abcam, ab42824). Immunoblot uncut membranes are provided in DATA S1.