Bace1

The hippocampus was isolated from the brain hemisphere and lysed in NETN lysis buffer containing protease inhibitor PMSF. Equal amounts of the lysates were loaded and separated using SDS–PAGE. Next, the proteins were transferred to nitrocellulose membranes (GE Healthcare) and blocked using 5% skim milk in TBST for 1 h. The membrane was incubated with indicative primary antibodies at 4°C overnight: GAPDH (1:1,000; Cell Signaling Technology), hAPP(1:1,000; Invitrogen), LAMP1 (1:1,000; Santa Cruz), and BACE1 (1:500; Santa Cruz). The membranes were then incubated with IRDye-conjugated anti-mouse or rabbit secondary antibodies for 2 h at RT. Proteins were detected using Odyssey CLx and quantified using the ImageJ software.

The protein expressions of the brain tissues and cell specimens were analyzed as described previously [25 (link)]. The primary antibodies included MRTF-A (1:1000, sc-398675, Santa Cruz, CA, USA), APP (#2452, Cell Signaling Technology, MA, USA), and BACE1 (1:1000, sc-33711, Santa Cruz, CA, USA), p62(1:1000, P0067, Sigma-Aldrich St. Louis, USA), Beclin1(1:1000, #3738, Cell Signaling Technology, MA, USA), mTOR (1:1000, ab32028, Abcam, MA, USA), p-mTOR(1:1000, sc-293133, Santa Cruz, CA, USA), and β-actin (1:1000, sc-8432, Santa Cruz, CA, USA). The antibodies were diluted in TBST buffer (50mM TrisHCl, 150mM NaCl, 0.1% Tween-20, pH 7.4) (P0015F, Beyotime, Shanghai, China) and incubated with the PVDF membrane at 4° C overnight. Corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, sc-2004, sc-2005, Santa Cruz, CA, USA) was subsequently incubated with the PVDF membrane for 90 minutes at room temperature. Signal detection was performed with an enhanced chemiluminescent (ECL) reagent (G2020-25ML, Servicbio, Shanghai, China). The luminescent signals were detected by a BioRad ChemiDoc MP system.