Timp 3

Cardiac tissue homogenates were lysed in NP-40 with protease inhibitor. Proteins (25 μg/well) were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with primary antibodies to EP4 (Santa Cruz Biotechnology, CA, USA), tissue inhibitor of metalloproteinase (TIMP)-3 (Proteintech, IL, USA), and GAPDH (Cell Signaling Technology, MA, USA) at 4 °C overnight. The membranes were then incubated with a secondary antibody (Cell Signaling Technology, MA, USA) for 1 h at room temperature and developed with ECL reagent. Enhanced chemiluminescence was detected with an LAS-3000 (Fujifilm, Tokyo, Japan).

After the MDA-MB-231 cells were treated with XLLXF (50, 100, and 200 µg/mL) for 24 and 48 h, total cell protein lysates were extracted using RIPA lysis buffer that contained protease and phosphatase inhibitor cocktails. Protein lysates (20 µg), which were determined by BCA analysis (Beyotime, China), were loaded onto 10% SDS-PAGE gels. The protein bands were transferred onto NC membranes and blocked with 5% non-fat milk for 1 h at room temperature. The NC membranes with proteins were incubated with diluted primary antibodies at 4 °C overnight. The primary antibodies used in the analyses were as follows: VEGFA (1:1,000, Proteintech), MMP2 (1:1000, Proteintech), MMP9 (1:1000, Cell Signaling Technology), Vimentin (1:1000, Proteintech), VE-cadherin (1:1,000, Cell Signaling Technology), TIMP-1 (1:1000, Proteintech), TIMP-3 (1:1,000, Proteintech), and Twist1 (1:1000, Proteintech). The membranes were incubated with relative sources of secondary antibodies (1:5000) at room temperature for 1 h. Specific protein bands were recognized with Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA). Image J software was used for image analysis.