Anti wnt16

Experimental cancer cells (after culturing in HUVEC-CM for 21 days) and respective control cells were lysed on ice in a lysis buffer (RIPA, Beyotime, Shanghai, China) with a protease inhibitor mixture cocktail (Roche, Switzerland). Western-blot analysis was performed by established protocols 12 . Anti-α-catenin, anti-E-cadherin, anti-ZEB-1, ZEB-2, Snail, Slug, anti-ZO-1 and anti-Laminin A1 primary antibodies were purchased from (Abcam); anti-MMP-1, -2, -3, -11, -12, -13, -17 and -21, anti- ITGA6, B1, B3, B4, B7, anti-FAK, P-FAK-Y397, anti-Src, P-Src-Y418, P-Src-Y529, anti-fibronectin, anti-Laminin B3, anti-Wnt-2, anti-Wnt-5B, anti-Wnt-16, anti-TGF-β, anti-β-catenin and anti-N-cadherin primary antibodies were purchased from (Epitomics); anti-Cdc-2, P-Cdc-2-Tyr15, CDK4, CDK6, Cyclin A, Cyclin D1, Cyclin D3, Cyclin E2, P15, P16, P21, P27, P53, Rb, P-Rb-S811, P-Rb-S780; anti-Bcl-xl, Bad, P-Bad-Ser112, Bak, Mcl-1, Puma and anti-β-actin were from (Cell Signaling Technology).

Following culture in MRC-5-CM for 14 days, whole liver cancer cells were lysed on ice in a lysis buffer (RIPA, Beyotime, Shanghai, China) with a protease inhibitor mixture cocktail (Roche, Switzerland). After centrifugation at 12000rpm for 30 minutes at 4℃, the protein concentrations of supernatants in samples were measured by the BCA protein assay (Thermo scientific, Rockford, IL, USA). Equal amounts of protein (50μg) were separated by 10%-12% NUPAGE Bis-tris Gel (Invitrogen, CA, USA) electrophoresis (constant voltage: 120mv) and transferred onto polyvinylidene fluoride (PVDF, 0.45μm) membranes (constant current: 350mA for 70/120 min). After being blocked by Tris-buffered saline and Tween 20 (TBST) buffer containing 5% non-fat powder milk for 2h, the membranes were incubated with primary antibodies overnight on ice. After washing the membranes several times in TBST while agitating, detection was performed using the appropriate secondary HRP-conjugated anti-mouse or anti-rabbit antibody. Immunoreactive bands on the blots were visualized with enhanced chemiluminescence reagent ECL kit (Beit Haemek, Israel). Anti-GAPDH, anti-WNT-2, anti-WNT-5B, anti-WNT16, anti-TGFB1, anti-CTNNB1, anti-IL6, anti-Nanog, anti-OCT4 and anti-CK19 primary antibodies were purchased from (Epitomics).