Rabbit anti human mmp2

Total protein was extracted from cells, and the concentration was determined using the BAC protein concentration assay kit (Beyotime Biotec, China). Samples were separated using 8–15% SDS-PAGE and then transferred to PVDF membranes (Millipore, NY, and the USA). Membranes were blocked for 1 h with 5% non-fat milk. Membranes were incubated with primary antibodies at 4°C. Antibodies used in this study were as follows: rabbit anti-human WNK3 (1:2,000; Abcam, UK), rabbit anti-human Cyclin D1 (1:2,000, Cell Signaling Tech), rabbit anti-human MMP-2 (1:2,000, Abcam), rabbit anti-human MMP-9 (1:2,000, Abcam), rabbit anti-human Snail1 (1:2,000, Abcam), rabbit anti-human E-cadherin (1:3,000, Abcam), rabbit anti-human Vimentin (1:3,000, Abcam), and mouse anti-human β-actin (1:2,000, Abcam). After three washes in TBST, the membranes were incubated with the appropriate secondary antibody (1:5,000, Abcam) in TBST for 2 h at room temperature. Proteins were detected using the ECL detection solution (Apexbio, Houston, USA).

CSps were stained according to a described protocol [11] (link), [26] (link). In brief, spheres were fixed in ethanol/acetone and stained in whole mount. The following primaries diluted in Dako saponin 0.1% (DAKO) were used: Rabbit anti human MMP2 (Abcam) and Rb anti human MMP9 (Abcam). Secondary antibodies conjugated with Alexa fluorochromes were used.