Atg5 2630

Cortex lysates from wild type and 2xTg-AD (APPswe/PSENldE9) mice were kindly provided by Dr Vilhelm A. Bohr (National Institutes of Health). Cellular protein was extracted with RIPA buffer (Beyotime, Shanghai, China) supplemented with complete protease inhibitor mixture (Roche, Mannheim). Subsequently, the western blot assay was performed by following the previous descriptions^{28 (link)}. The antibodies used include: LC3B (L7543), Flag (F7425), and β-actin (A1978) from Sigma-Aldrich (St. Louis, MO, USA); ATG5 (#2630) and ATG7 (#8558) from Cell Signaling technology (San Diego, USA), p62/SQSTM1 (18420-1-AP), GFP (50430-2-AP), cathepsin D (CTSD) (21327-1-AP), and ERβ (14007-1-AP) from Proteintech (Wuhan, China); Lysosome-associated membrane protein type 2 (LAMP2) (A14017) and BACE1 (A5266) from Abclonal (Wuhan, China); α7nAChR (501588) from ZENBIO (Chengdu, China); peroxidase-conjugated immunopure goat anti-rabbit and anti-mouse IgG (HL) from Abclonal. Enhanced chemiluminescence horseradish peroxidase was used to visualize protein bands. NIH ImageJ software was used to measure the intensity of the bands.

Lysate proteins (20 μg) from treated cells were subjected to Western blot analysis following the protocols previously described [27 (link),28 (link)]. Briefly, the primary antibodies used include phosphor-Thr¹⁷² AMPKα (AMPK-p, 2535), total AMPKα (AMPK-T, 2603), and Atg5 (2630) from Cell Signaling Technology (Danvers, MA, USA); LC3 (L7543), Flag (F7425), and β-actin (A5441) from Sigma-Aldrich (St Louis, MO, USA); Beclin 1 (SC11427), pERK (SC-7383), and t-ERK (SC94) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and the secondary antibodies were horseradish peroxidase-coupled goat anti-mouse-IgG (AP124P) from Merck Millipore (Darmstadt, Germany) and goat anti-rabbit-IgG (81–6120) from Invitrogen Corporation (Camarillo, CA, USA).

Pancreatic cancer cell lines (AsPC-1, BxPC-3, PANC-1, CFPAC-1 and SW1990) were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂. Antibodies against YAP (#14074) and ATG5 (#2630) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-active YAP (ab205270) antibody was purchased from Abcam (Cambridge, MA, USA). Antibodies against p62 (sc-28359) and GAPDH (sc-32233) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-LC3 (NB100-2220) antibody was obtained from Novus Biologicals (Littleton, CO, USA). Anti-Flag antibody (F3165), cycloheximide (CHX, 01810), verteporfin (SML0534) and chloroquine (CQ, C6628) were obtained from Sigma-Aldrich (St.Louis, MO, USA).