Af1552

Cells and tissues were homogenized for 25 min on ice in RIPA lysis buffer, supplemented with protease inhibitors, phosphatase inhibitors, and 100 mM PMSF (KeyGEN BioTECH, Shanghai, China). Extracted proteins were resolved by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany) using a semi-dry apparatus. Membranes were blocked in 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1h at room temperature, followed by overnight incubation with antibodies targeting CXCL12 (1:1000, 3530, Cell Signaling Technology), CXCR4 (1:1000, 11073-1-AP, Proteintech), Wnt5a (1:1000, ab174963, Abcam), β-Catenin (1:5000, ab174963, Abcam), E-cadherin (1:5000, AF1552, Beyotime), N-cadherin (1:2000, AF0243, Beyotime), Vimentin(1:5000, AF0218, Beyotime) and GAPDH (1:2000, ab181602, Abcam) at 4 °C. After washing three times with TBST for 10 min at room temperature, membranes were incubated with secondary antibodies for 1 h, and washed three times with TBST. Positive bands were visualized using an ECL Plus kit (Beyotime, China).

Western blot analysis was performed to detect protein expression levels. Proteins were extracted from cell lysates using a cell lysis buffer (Beyotime, Shanghai, China) and separated by 10% SDS-PAGE followed by electrotransfer onto nitrocellulose membranes, blocking with skimmed milk at room temperature for 1 h. Membranes were incubated overnight at 4°C with primary antibodies, including anti-Nectin2 (ab135246; Abcam, Cambridge, USA), anti-GAPDH (AF1186; Beyotime), anti-β-actin (ab8226; Abcam), anti-MMP2 (AF1420; Beyotime), anti-MMP9 (AF5234; Beyotime), anti-cleaved caspase-3 (ab32042; Abcam), anti-bax (bs-0127R; Bioss, Beijing, China), anti-bcl2 (AF6285; Beyotime), anti-N-cadherin (AF0243; Beyotime), anti-E-cadherin (AF1552; Beyotime) and anti-ANXA2 (AF5115; Beyotime). Membranes were washed several times with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (KC5G5; Aksomics, Shanghai, China) at room temperature for 1 h. Protein blots were visualized using an ECL kit (P0018FS; Beyotime), and protein levels are expressed as the ratio of band optical intensity relative to that of GAPDH. Densitometry analyses were performed using ImageJ software (version 1.52; NIH, Bethesda, USA).