Anti beclin1 antibody

For IHC, the left CA1 region of random 24 rats from each group was fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. After being rehydrated, the samples were immersed with 3% H₂O₂ at room temperature for 10 min to block endogenous horseradish peroxidase (HRP) activity, and antigen retrieval was performed by microwave for 8 min in citrate buffer. Each section was incubated with normal goat serum in PBS for 30 min at room temperature and then incubated with primary antibody (1 : 100; anti-Beclin1 antibody, Abcam; anti-LC3 antibody, Abcam) at 4°C overnight. After phosphate-buffered saline (PBS) washing, the slides were then incubated with a corresponding second antibody (Abcam) at room temperature for 30 min and stained with diaminobenzidine (DAB; ChemMate TM DAKO Envision TM Detection Kit, DAKO). The slides were then counterstained with hematoxylin, dehydrated, and mounted. The degree of staining was controlled by microscopic observation (Olympus) with 200x magnification. The IHC scores were calculated by the software ImageJ which showed percent contribution of different grades of positive.

Human pancreatic cancer cell lines BxPc-3, PANC-1, SW1990, AsPC-1, and MiaPaCa-2 were obtained from and validated by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured as per their instructions. Briefly, cells were cultured with a humidified atmosphere of 95% air and 5% CO₂ at 37°C in Dulbecco's modified Eagle's medium (DMEM) (HyClone, Logan, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) added with 100 μg/mL ampicillin and 100 μg/mL streptomycin. In experiments designed to elevate the level of intracellular ROS, H₂O₂ was added in the serum-free media with a final concentration of 100 μmol/L for 24 h. Different autophagic inhibitors were used in this study: 3-MA of 5 mmol/L or CQ of 40 μmol/L for 24 h. Antibodies were obtained from the following resources: anti-β-actin antibody (Santa Cruz, USA), anti-Nrf2 antibody (Abcam, USA), anti-Beclin1 antibody (Abcam, USA), anti-LC3 antibody (Sigma, USA), and anti-p62 antibody (Proteintech Group, USA). Autophagic inhibitors were obtained from the following resources: 3-MA (HanBio, China) and CQ (Sigma, USA).

Protein samples were extracted from tissues and cells with the same procedures described previously. Briefly, the heart tissues and myocardial cells were lysed in RIPA buffer and then centrifuged at 4°C 12 000 g for 15 minutes. The supernatant was collected, and protein concentrations were determined by bicinchoninic acid (BCA) protein assay (Beyotime, Shanghai, China). Protein extracts were separated by SDS‐PAGE and transferred onto a nitrocellulose membrane. Then, the membranes were blocked and incubated with the following primary antibodies: anti‐BNP antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐β‐MHC antibody (1:2000, Sigma, St Louis, MO, USA), anti‐TRPV3 antibody (1:200, Abcam, Cambridge, MA, USA), anti‐Beclin‐1 antibody (1:500, Abcam), anti‐LC3‐II antibody (1:500, Abcam)and anti‐β‐actin (1:2000, Santa Cruz Biotechnology). β‐actin was used as a loading control. After incubation at 4°C overnight, membranes were washed for three times with Tris Buffered Saline with Tween‐20 (TBST), 10 minutes each time, then incubated with secondary antibody for 1 hour at room temperature in the dark. Finally, the membranes were washed for three times with TBST, 10 minutes each time, and scanned by Odyssey Imaging System (LI‐COR Biosciences, Lincoln, NE, USA).

The SH-SY5Y cell line of human neuroblastoma was collected from the Stem Cell Bank of the Chinese Academy of Sciences. Semaglutide (peptide purity: 95.77%) and liraglutide (peptide purity: 95.77%) were obtained from Synpeptide Co. (Shanghai, China); 6-OHDA and the anti-LC3-II antibody were obtained from Sigma-Aldrich (St. Louis, MO, USA); the anti-P62 antibody, anti- Atg7 antibody, and anti-beclin1 antibody were obtained from Abcam (Cambridge, UK); and the β-actin antibody was obtained from Bioworld Technology Co. (Shanghai, China). The reactive oxygen species (ROS) assay kit and mitochondrial membrane potential assay kit with JC-1 were purchased from Nanjing KeyGen Biotechnology Company (Nanjing, China).

NP cells were harvested and washed three times with PBS and fixed with 4% formaldehyde for 15 min at room temperature after transfection with miR-129-5p mimic or inhibitor. Nonspecific binding sites were blocked by incubation with goat serum for 30 min. Cells were incubated overnight at 4°C with anti-Beclin-1 antibody (1:150; Abcam, Cambridge, UK), followed by a 1-h incubation with Cy3-conjugated goat anti-rabbit IgG (GE Healthcare, Little Chalfont, UK; 1:100 dilution) after three washes with PBS at room temperature. Cells were incubated with 4′,6′-diamidino-2-phenylindole (Beyotime, Beijing, China), washed three times, and examined by fluorescence microscopy (Olympus, Tokyo, Japan).
Cells transfected with miR-129-5P mimic or inhibitor were incubated with goat serum for 30 min, followed by overnight incubation at 4°C with primary antibody against LC3 (1:150; Abcam) and a 1-h incubation with secondary antibody (1:100; GE Healthcare). Samples were washed three times with PBS at room temperature, and images were captured on an epifluorescence microscope (Olympus). To quantify autophagy, the percentage of cells with punctuate red fluorescent protein (RFP)-LC3 fluorescence was counted. Cells with more than 10 punctae were considered as undergoing autophagy.