Horseradish peroxidase conjugated secondary antibody

The total cellular protein was extracted using M-PER Mammalian Protein Extaction Reagent supplemented with 1% protease and phosphatase inhitor (Thermo Scientific, USA). Proteins were separated by SDS-PAGE, transferred to PVDF membranes (Bio-Rad, USA) and blocked with 5% non-fat milk in TBST buffer (TBS-Tween 20). Then membranes were incubated with primary antibodies overnight at 4°C. After washing 3 times with TBST, membranes were treated with horseradish peroxidase-conjugated secondary antibodies (Huabio, China) for 1 hour at room temperature and probed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, USA).

Whole cells were homogenized in protein lysis buffer (P0013; Beyotime Biotechnology, Shanghai, China) after treatment with indicated compounds, and protein concentrations in the lysates were measured using a BCA Protein Assay kit (P0010; Beyotime Biotechnology). An equal amount of protein was fractionated by SDS-PAGE, and then transferred onto a PVDF membrane (Millipore, Billerica, USA). After being blocked with 5% fat-free milk for 1 h, membranes were incubated with primary antibodies at 4°C overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Huabio, Hangzhou, China) for 1 h at room temperature and detected using ECL reagent (34095; Thermo Fisher Scientific, Waltham, USA).

Proteins were removed from rat aortic SMCs following lysis and after intervention. The cells were divided into four groups, as described, and 40 µg of protein per lane was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Epizyme, Beijing, China), transferred to a polyvinylidene difluoride membrane (Millipore Sigma, Billerica, MA, USA), and blocked with western quick-blocking buffer (Beyotime) for 15 min at room temperature. The blocked membranes were then incubated overnight with primary antibodies, namely goat anti-SM22α (1:500; Abcam, Cambridge, UK), rabbit anti-OPN (1:1000; Abcam), and rabbit anti-MMP9 (1:1000; Abcam). After 12 h to 14 h, the membranes were incubated with a horseradish-peroxidase-conjugated secondary antibody (HuaBio, Hangzhou, China) for 1 h at room temperature. The immunoblots were probed using an enhanced chemiluminescence substrate (Thermo Fisher Scientific), and an imaging system (Bio-Rad Laboratories, Hercules, CA, USA) was used for blot detection and recoding of chemiluminescence. The results were normalized to that of GAPDH, and experiments were performed in triplicate.