Bicinchoninic acid assay kit

After reaching 80–90% confluence, the cells in culture plates were lysed using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with 1 mM phenylmethylsulfonyl fluoride protease inhibitor (Beyotime Institute of Biotechnology). The concentrations of proteins were determined using the bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). The proteins were boiled following addition of the sample loading buffer and the denatured proteins were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes, which were blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology), as previously described (20 (link)). Subsequently, the membranes were incubated with primary antibodies against VEGF (cat. no. ab106041; 1:1,000 dilution; Abcam, Cambridge, MA, USA) and β-actin (cat. no. ab106045; 1:5,000 dilution; Abcam), followed by incubation with the secondary antibody (cat. no. ab6721; 1:5,000 dilution; Abcam). The bands of proteins were visualized using enhanced chemiluminesence (EMD Millipore, Billerica, MA, USA) and detected with the ChemiDoc MP imager (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Protein amounts in cell lysates were measured using a bicinchoninic acid assay kit (Beyotime, Beijing, China). Equal amounts of total protein were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). The blocked membranes were incubated with primary antibodies targeting VCAM-1, ICAM-1, E-selectin, β-actin (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), NF-κB p65, phosphorylated inhibitor of NF-κB (IκB)-α, and IκB-α (1:1000, Cell Signaling Technology, Danvers, MA, USA) for 1 hour. After washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (1:3000, MultiSciences, Hangzhou, China) for 1 hour. The blots were developed using enhanced chemiluminescence substrate (Thermo, Waltham, MA, USA) and X-ray film. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to quantitate protein bands.

Heart tissues were homogenized in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). The homogenates were centrifuged at 1,600 × g for 10 min at 4°C. A bicinchoninic acid assay kit (Beyotime Institute of Biotechnology) was used for protein quantification. A total of 20 µg protein was electrophoresed on 10–15% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were subsequently blocked with 5% (w/v) non-fat milk in TBS containing 0.1% (v/v) Tween-20 and incubated overnight at 4°C with anti-PPARα (cat no. WL00978; 1:1,000; Wanleibio, Co., Ltd., Shanghai, China), anti-Desmin (cat no. ab32362; 1:8,000; Abcam, Cambridge, UK), anti-GRP78 (cat no. WL00621; 1:1,000; Wanleibio, Co., Ltd.), and anti-GADPH (cat no. ab181602; 1:8,000; Abcam). Following washing, bound antibodies were detected following incubation for 1 h at room temperature with peroxidase-conjugated goat anti-rabbit IgG (cat no. ZB-2301; 1:10,000; OriGene Technologies, Inc., Beijing, China). Blots were developed using Western Lightning BeyoECL Plus reagent (Beyotime Institute of Biotechnology) and were quantified using ImageJ software (version 2.1.4.7; National Institutes of Health, Bethesda, MD, USA).

Total protein was extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Total protein was quantified using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). Subsequently, 15 µg protein/lane was separated using 8–10% SDS-PAGE and transferred onto a PVDF membrane, which was subsequently blocked with 5% skimmed milk diluted with TBS-1% Tween (TBST) for 2 h at room temperature. The PVDF membrane was then incubated overnight at 4°C with polyclonal antibodies for MMP-2 (cat. no. ab215986; 1:1,000; Abcam) and MMP-9 (cat. no. ab219372; 1:1,000; Abcam), with GAPDH antibody (cat. no. ab8245; 1:10,000; Abcam) serving as an internal control. The membrane was further incubated with horseradish peroxidase-conjugated anti-rabbit (cat. no. sc-2030) or anti-mouse (cat. no. sc-2005) immunoglobulin-G secondary antibodies (1:5,000; Santa Cruz Biotechnology, Inc.) diluted with TBST for 1 h at room temperature. The resulting protein signals were detected using an enhanced chemiluminescence reaction system (EMD Millipore) and quantified using ImageJ software (version 1.8.0; National Institutes of Health).

Protein extracts were obtained using the Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology), total protein concentration was quantified using the bicinchoninic acid assay kit (cat. no. p0012s; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. A total of 80 µg of protein was loaded per lane, then separated via SDS-PAGE on a 10% of gel. The separated proteins were transferred to polyvinylidene-difluoride membranes (EMD Millipore). After blocking for 2 h at 25°C in 5% skimmed milk, the membranes were incubated with primary antibodies anti-Nrf2 (1:1,000), anti-histone 3 (1:1,000), anti-β-actin (1:3,000) and anti-NFκB (1:1,000) overnight at 4°C. The membranes were then washed with Tris-buffered saline and Polysorbate 20 and incubated with secondary antibodies (1:10,000) for 1 h at room temperature. The protein bands were visualized using enhanced chemiluminescence (cat. no. P0018AS; Beyotime Institute of Biotechnology) and the ChemiDicTM XRS+ Imaging System (Bio-Rad Laboratories, Inc.), and the densities of the immunoreactive bands were analysed using Image J software (National Institutes of Health). β-actin was used as the loading control for quantification.

HBMSCs were lysed using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) on ice for 30 min and supernatants were acquired though centrifugation at 14,000 × g for 20 min at 4°C. Subsequently, proteins were quantified using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology) and 50–100 µg protein was resolved by 8–10% SDS-PAGE and transferred to a polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking with 5% skim milk powder in TBS-0.1% Tween-20 (TBST) for 1 h at 37°C, membranes were incubated overnight at 4°C with the following primary antibodies: Anti-TGF-β (cat. no. 3709; 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA); anti-p-Smad2 (cat. no. 8828; 1:1,000; Cell Signaling Technology, Inc.); anti-COL4A1 (cat. no. sc-517572; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-GAPDH (cat. no. 3683; 1:5,000; Cell Signaling Technology, Inc.). Membranes were washed with TBST and incubated with horseradish peroxidase-conjugated anti-rabbit or mouse IgG secondary antibody (cat. nos. 7076 and 7074; 1:5,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. The membranes were visualized with BeyoECL Plus (Beyotime Institute of Biotechnology, Haimen, China) and analyzed using ImageJ 2× software (National Institutes of Health, Bethesda, MD, USA).