Matrix metalloproteinase mmp 2

Cells were harvested and lysed with ice-cold lysis buffer (Sigma, USA) and protein concentration determined using a protein assay kit (Bio-Rad Laboratories, Hercules, USA). Denatured proteins (100 μg) were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to Hybond membranes (Amersham, Munich, Germany), and blocked overnight in 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST). For immunoblotting, the membrane was incubated with antibodies against E2F-1, RhoC (1:300, Santa Cruz Biotechnology, Santa Cruz, USA), p53, caspase 3, stat3, Bcl-2, cyclin D1, matrix metalloproteinase (MMP) 2 and MMP9 (1:300, Bioss,BeiJing, China), HuR(proteintech, Chicago, USA). The membranes were then rinsed with TBST and incubated with anti-mouse or anti-rabbit IgG antibodies conjugated to horseradish peroxidase (1:5000; Dako, Carpinteria, USA) for 2 h. Bands were visualized on X-ray film (Fuji film, Tokyo, Japan) using Image Quant LAS 4000 (Fuji film) and ECL Plus detection reagents (Santa Cruz Biotechnology). β-actin (ZSGB-Bio,BeiJing, China) was used as a loading control.

Total proteins were extracted from atrial samples using radioimmunoprecipitation assay buffer plus phosphoprotease inhibitors (ASPEN Biotechnology, China). The same amount (40 μg) of extracted protein was separated by electrophoresis in SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% blocking buffer for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against CD63 (Biorbyt, England), CD81 (Abcam, USA), TSG101 (Sigma-Aldrich, Germany), Rab27a (Sanying Biotechnology, China), collagen I (Novusbio, USA), collagen III (Abcam, USA), matrix metalloproteinase (MMP)-2 (Bioss, USA), MMP-9 (Bioss, USA), tissue inhibitor of metalloproteinase 3(TIMP3) (Lsbio, USA), and transforming growth factor-β1 (TGF-β1) (Sanying Biotechnology, China). The membranes were washed three times with tris-buffered saline with 0.1% Tween® 20 and then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (ASPEN Biotechnology, China) for 1 h at room temperature. The blots were exposed with an ECL Detection Kit (ASPEN Biotechnology, China). The expression levels of the proteins were determined and normalized to the relative intensity of GAPDH using image analyzer software (AlphaEase FC, USA).