Anti mmp2

Cell samples were lysed in NP40‐containing buffer with PMSF, protease inhibitors, and phosphatase inhibitors. Individual cell lysate samples (30 µg/lane) were separated via SDS‐PAGE on a 12% resolving gel and electro‐transferred to polyvinylidene difluoride membranes (Millipore, Hong Kong). The membranes were blocked with 5% non‐fat dry milk in TBST and incubated overnight at 4°C with primary antibodies, including anti‐human LIPH (1:500, Proteintech, Wuhan, China), anti‐GAPDH (Cell Signaling Technology, Danvers, MA), anti‐CD44 (60224‐1‐Ig), anti‐CD24 (10600‐1‐AP), anti‐Oct4 (11263‐1‐AP), anti‐Sox2 (11064‐1‐AP), anti‐vimentin (WL01960), anti‐MMP2 (WL01579a, WanleiBio, China), anti‐FAK p397 (ab24781, Abcam, UK), anti‐Integrin‐ß3 (WL02735b, WanleiBio), anti‐p‐AKT (WLp001, WanleiBio), anti‐AKT (WL0003b, WanleiBio), anti‐RAS (WL0257, WanleiBio), anti‐CAPN2 (abs137284), anti‐Paxillin (WL00415, WanleiBio), anti‐Vinculin (abs131199), and anti‐Tubulin‐α (2144s, CST, USA). The bound antibodies were detected with horseradish peroxidase (HRP)‐conjugated secondary antibodies (1:10 000). The immunoblotting signals were visualized using enhanced chemiluminescent reagents. The ratios of target proteins to control GAPDH were determined by densitometric analysis using the ImageJ software application (NIH, Bethesda, MD, USA).

Cells were harvested for 48 h after transfection. Radioimmunoprecipitation assay buffer was added to the samples and the samples placed on ice for 5 min. The samples were then centrifuged at 12,000 rpm, 4 °C for 10 min and the supernatant removed to obtain total protein. After total protein was quantified, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was carried out at 80V for 2.5 h. After being blocked for 1 h with 5% skim milk, the membranes were incubated with specific primary antibodies overnight at 4 °C followed by incubation with the secondary antibodies for 2 h at room temperature. The anti-SPOCK2 polyclonal antibody (1:200) was purchased from Santa Cruz, CA, USA, the anti-MT1-MMP (1:1,000) was purchased from Proteintech, Wuhan, China, and the anti-MMP2 (1:500) was purchased from Wanleibio, Shenyang, China. Secondary antibody (1:5,000) was purchased from Wanleibio, Shenyang, China. The film was scanned, and the optical density value of the target strip analyzed using a gel image processing system (Gel-Pro-Analyzer software). All assays were performed in triplicate.