As1004

Western blot analysis was performed according to a previously described method.32 (link) The total protein was extracted from ovarian tissue using radioimmunoprecipitation assay buffer (ASPEN, AS1004, Wuhan, Hubei, People’s Republic of China), and the protein lysate was separated on 10% polyacrylamide gels and then transferred to polyvinylidene difluoride (PVDF) membranes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal loading control. The blots were visualized with enhanced chemiluminescence (ECL) reagents. The quantitative analysis of each band was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated three times and the results were expressed as the average ± standard deviation (SD).

Tissues or cells were lysed via a lysis buffer (#AS1004, Aspen, South Africa) with 1% protease inhibitor (#AS1008, Aspen, South Africa) on ice. First, the protein fractions were collected, after which SDS-PAGE separation was carried out, followed by transfer onto a NC membrane (#IPVH00010, Millipore, USA). And then membranes were blocked by using 5% skim milk and probed at 4°C overnight, followed by HRP-conjugated secondary antibodies (#AS1058, Aspen, South Africa). Protein was visualized using a chemiluminescence detection system (#LiDE110, Canon, Japan). Antibodies were as follows: anti-Bcl-2 (#MAB-8272, 1: 500, R&D System, USA), anti-Bax (#AF820, 1: 1000, R&D System, USA), anti-cyclin D1 (#AF4196, 1: 500, R&D System, USA), and anti-cyclin D3 (#MAB6570, 1: 500, R&D System, USA). All experiments were conducted in triplicate.

Following transfection for 48 h, total proteins from HepG2 cells and HepG2.2.15 cells were extracted using RIPA buffer (AS1004; ASPEN) and evaluated using the BCA protein assay kit (AS1086; ASPEN). Next, the proteins were separated using SDSPAGE and transferred onto PVDF membranes (IPVH00010; Millipore, Burlington, MA, USA). After blocking with 5% skim milk in PBST for 1.5 h, the membranes were incubated with primary antibodies against beclin-1 (cat. no. #3738; 1:2000 dilution; Cell Signaling Technology, Danvers, MA, USA), p62 (cat. no. ab109012; 1:2000 dilution; Abcam), and LC3I/II (cat. no. #4108; 1: 1000 dilution; Cell Signaling Technology), ATG7 (cat. no. ab133528; 1:1000 dilution; Abcam), or β-actin (cat. No. TDY051; 1:10000 dilution; Beijing TDY Biotech Co., Ltd., Beijing, China) overnight at 4˚C. The membranes were then washed and incubated with a secondary antibody for 1 h. Finally, the target protein bands were examined using an ECL detection system reagent (ASPEN, AS1059) according to the manufacturer’s protocol.

Fifty μg of protein was extracted from the ischemic myocardial tissues at 4 weeks after MI. The tissues were lysed in radioimmunoprecipitation assay (AS1004, Aspen, Wuhan, China), and lysis buffer and protein concentrations were determined using the BCA kit (AS1086, Aspen). The proteins were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The membranes were blocked by incubation with 5% bovine serum albumin in a TBS-Tween buffer (10 mM Tris-HCl, 150 mM NaCl, and 0.5% Tween-20) for 1 hour at room temperature, and subsequently, incubated with different primary antibodies: rabbit anti-TH (Santa, Shanghai, China), anti-GAP43 (1:1,000; Abcam, Cambridge, UK), anti-NGF (1:500; Abcam), anti-CHAT (1:500; Bioss, Beijing, China), anti-VACHT (1:500; Abcam), or anti-GAPDH (1:10,000; Abcam) overnight at 4°C. After the membrane was washed three times, the blots were incubated with secondary HRP conjugated goat anti-rabbit antibody (1:10,000; Pierce, Rockford, IL, USA) for 30 minutes at room temperature. Membranes were detected by enhanced chemiluminescence (Beyotime Biotechnology, Jiangsu, China) and exposed to film in the dark. The optical density intensity of each band was measured using AlphaEaseFC software (Alpha Innotech Corp., San Leandro, CA, USA). Results are shown as the optical density ratio to GAPDH.