β actin

Cells were washed twice with ice-cold PBS and scraped into 1 ml of RIPA lysis buffer (Sangon Biotech Co., Ltd., Shanghai, China) with protease inhibitors of PMSF. Lysates were clarified by centrifugation (12,000 rpm for 10 min) and the supernatants were collected. Equal amounts of protein (50 µg) underwent SDS-PAGE and then were electrotransferred to PVDF membranes (Millipore Corp., Billerica, MA, USA), which were then sealed at room temperature for 2 h. The membranes were then incubated overnight at 4°C with the following primary antibodies diluted in blocking buffer: SCD1 (catalog sc-14715), and CD36 (catalog sc-7309) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and the internal control β-actin (Cell Signaling Technology, Inc. Danvers, MA, USA). Then, the membranes were washed and incubated with the appropriate HRP-conjugated secondary antibody (Beijing CoWin Bioscience Co., Ltd., Beijing, China) in PBST for 2 h at room temperature. The membranes were then washed three times and reacted with chemiluminescent agent for 5 min. Then they were ECL labeled, exposed, and displayed. Quantification of the resulting images was performed by densitometry with Gel-Pro Analyzer 4.0 software (Media Cybernetics, Inc., Bethesda, MD, USA) and the final readings were normalized against β-actin.

The proteins were extracted from the myocardial tissue samples and analyzed by western blotting, as described previously.^{20 (link)} Extracted protein lysates from the left ventricle were separated on SDS-PAGE and then transferred to the PVDF (polyvinylidene fluoride) membrane. After sealing with 5% (m/v) skimmed milk powder, the membranes were then incubated with primary antibodies, including ADAM17 (Abcam, AB39162, 1 : 2000), TIMP-3 (Abcam, AB3984, 1 : 2000) and β-actin (Wanleibio, WL01845, 1 : 1000) at 4 °C overnight and subsequently with sheep anti-rabbit IgG-HRP (Wanleibio, WLA023, 1 : 5000) at room temperature for 45 minutes. The blotting band density was quantified by densitometric analysis using Image pro-Plus 6.0 software (Media Cybernetics, USA) and then normalized to the values of β-actin.

RIPA lysis buffer (Boster, Wuhan, China) was used to extract protein from indicated cells. BCA Protein Assay Kit (Thermo Scientific, USA) was used to measure protein concentration. A total of 60 μg of protein was separated on 10% SDS-PAGE gels and blotted onto 0.22 μm nitrocellulose membranes (Boster). The membranes were blocked for 2 hours with 5% nonfat dry milk diluted with tris‑buffered saline (TBS) and incubated with primary antibodies (rabbit monoclonal anti-OLFM4 (1:2,000), rabbit monoclonal anti-phosphorylated-FAK antibody (1:500), rabbit monoclonal anti-FAK antibody (1:1,000), rabbit monoclonal anti-MMP2 (1:1,000), rabbit monoclonal anti-MMP9 (1:3,000), and mouse monoclonal anti-β-actin (1:3,000) (Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4℃. The membranes were washed with tris-buffered saline containing 0.1% Tween20 (TBST), and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, 1:3,000; goat anti‑mouse, 1:2,000; Wuhan Boster) for 1 h at 37℃. Enhanced chemiluminescence reagent (Merck Millipore, Germany) was used to detect the signal on the membrane. The data was analyzed via densitometry using Image-Pro plus 6.0 software (Media Cybernetics, Rockville, MD, USA) and normalized to the expression of the internal control (β-actin).

Protein samples were prepared from NP tissues using radioimmunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology, Ltd.). Protein concentration was measured using a bicinchoninic acid Protein assay kit (Wuhan Boster Biological Technology, Ltd.). Protein samples (20 µg) were then separated using 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (EMD Millipore). The membrane was subsequently blocked with 5% skimmed milk powder for 1 h at room temperature and incubated with primary anti-nuclear factor (NF)-κBp65 (cat. no. 8242) and β-actin (cat. no. 4970) antibodies (Cell Signaling Technology, Inc.) at a dilution of 1:1,000 overnight at 4°C. Samples were then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibodies (cat. no. 7074; 1:5,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. Protein bands were visualized using an ECL chemiluminescence kit (EMD Millipore). Protein levels were calculated relative to β-actin and Image-ProPlus software (version 6.0; Media Cybernetics, Inc.) was used for densitometry analysis.

The expression of the apoptosis-related proteins (Bcl-2 and Bax) and AMPK/Sirt1 were determined using Western blotting. Briefly, equivalent protein samples from each group were separated using 10% or 12% SDS-PAGE and then transferred onto nitrocellulose membranes. The membranes were blocked using 5% skim milk at RT for 1 h and incubated with primary antibodies against p-AMPKα (Thr172) (no. 2535, 1:1000, Cell Signaling Technologies, MA, USA), Bcl-2 (no. 26539-1-AP, 1:2000, Proteintech, Wuhan, Hubei, China), AMPKα (no. 2603, 1:1000, Cell Signaling Technologies, MA, USA), Bax (no. 2772, 1:1000, Cell Signaling Technologies, MA, USA), Sirt1 (no. 9475, 1:1000, Cell Signaling Technologies, MA, USA), and β-actin (no. TA09, 1:2000, ZSGB-BIO, Beijing, China) at 4 °C overnight. The membranes were rinsed thrice with Tris-buffer saline with 0.25% Tween-20 (v/v) and incubated with a horse radish peroxidase (HRP)-conjugated secondary antibody (no. BA1054, BA1050, 1:30000, Boster Biological Technology, Wuhan, China). The membranes were visualized using a SuperSignal™ West Dura Extended Duration Substrate kit (Thermo Fisher Scientific, IL, USA). After exposing the X-ray film, the density of the bands was standardized over β-actin and calculated using a Gel-Pro Image Analysis software (Media Cybernetics, Rockville, MD, USA).