P tau ser396

The tissue and cell samples were collected and lysed with RIPA buffer (Beyotime, Beijing, China) containing 1% phenylmethylsulphonyl fluoride (PMSF) (Beyotime, Beijing, China). The protein concentration was measured by BCA Protein Assay Kit (Beyotime, Beijing, China). Primary antibodies for calcium voltage-gated channel subunit alpha-1 C (CACNA1C) (1: 200) (Santa Cruz, Santa Cruz, CA, USA), phosphorylated-tau (p-tau) (Ser 202) (1: 400) (Boster, Wuhan, China), p-tau (Ser 396) (1: 5000) (Abcam, Cambrisge, MA, USA), p-tau (Ser 404) (1: 500) (Sangon, Shanghai, China), tau (1: 500) (Sangon, China) and amyloid-β (Aβ) (1: 1000) (Sigma, St. Louis, USA) were used in this study.
Equal amounts of protein samples were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% dried skimmed milk powder and incubated with primary antibodies and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H+L) secondary antibody (1: 5000) (Beyotime, Beijing, China). Proteins were visualized by ECL Reagent (Seven Sea Biotech, Shanghai, China) and imaged with a gel imaging system. β-actin was used as an internal control.