Mouse anti collagen 3

Western blot was performed to determine the levels of collagen I, collagen III, TIMP-1/2/3, and MMP-13 in rat myocardium. The myocardium was ground into a homogenate with RIPA lysis buffer (the mass volume ratio 1:10 (mg/μL)), and then the homogenate was centrifuged for 15 min at 4°C to separate the supernatant [28 (link)], and the protein concentration was detected by BCA Kit (Beijing Biyuntian Co., China). The same amount of protein was separated by boiling with 10% SDS-PAGE. The protein gels were then removed to the nitrocellulose membrane by electrophoresis (Amersham, USA). The protein bands were incubated with the primary antibodies for mouse anti-collagen III (1:500; Sigma, USA), MMP-13 (1:400; Sigma, USA), GAPDH (1:2,000; Kangcheng, China), rabbit anti-collagen I (1:1,000; Abcam, USA), TIMP-1 (1:500; Abcam, USA), TIMP-2 (1:1,000; Abcam, USA), and TIMP-3 (1:1,000; CST, USA). The bands were then washed and incubated with the corresponding secondary antibodies (1:3,000; CST, USA) for 1 h. Bands were visualized on a FluorChem M MultiFluor System (ProteinSimple, USA) using chemiluminescence detection reagents. Optical density analysis was done with AlphaEaseFC (Alpha, USA).

MCF were harvested and lysed in a lysis buffer. Western blots were performed as previously described [14 (link)]. Equal amount of proteins from each sample were subjected to SDS-PAGE, and then transferred to nitrocellulose membranes. The membranes were blocked with 5 % BSA and incubated at 4 °C overnight with specific primary antibodies: rabbit anti-PCNA (diluted 1:100), rabbit His-tag (diluted 1:100), goat anti-collagen I (diluted 1:200), mouse anti-α-SMA (diluted 1:100), rabbit anti-TGF-β1 (diluted 1:200), goat anti-gp91^phox (diluted 1:200) (Santa Cruz Biotechnology, USA), or mouse anti-collagen III (diluted 1:200) (Sigma, USA). The horseradish peroxidase-conjugated second antibodies (diluted 1:5000) (Zhongshan Biotechnology, China) were incubated for 2 h at room temperature. Proteins were detected by ECL detection system.