Ve cadherin

Cells were lysed in RIPA lysis buffer containing protease inhibitor cocktail (Roche, Basel, Switzerland). The protein concentration was determined with a BCA kit (Pierce, Rockford, IL, USA). Equal protein was separated with 12% SDS–PAGE gel and electrotransferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat dry milk in TBST for 2 h. The membrane was incubated with poFUT1 (1:1000, Proteintech, Wuhan, China), VE-cadherin (1:500, Elabscience, China), CD31 (1:1000, Proteintech), p-LIMK (1:1000, Abcam), LIMK (1:1000, Abcam), p-cofilin (1:1000, Abcam), cofilin (1:1000, Abcam), Rho A-GTP (1:2000, Abcam), and Rho A (1:500, Abcam) antibody overnight at 4 °C. After washing with TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:3000, Beyotime, Shanghai, China) for 1 h at room temperature. An enhanced chemiluminescence (ECL) detection system (Bio-Rad) was used to visualize immunoreactivity bands. The relative protein level was quantified by densitometry and normalized to GAPDH level using Image J software (NIH, Bethesda, MD, USA).

Recombinant human IL-1β (HZ-1164) was purchased from Proteintech Group (Wuhan, China). Cell counting kit-8 (CCK-8) was purchased from Beyotime Biotechnology (Shanghai, China). Matrigel was obtained from Corning Inc. USA. p38 inhibitor (SB203580) and PI3K inhibitor (GDC-0941) were acquired from Selleck Chemicals (Shanghai, China). Antibodies:VEGFR-1(#13687-1-AP), p38 (#14064-1-AP), MMP-9 (#10375), MMP-2 (#10373-2-AP), GAPDH (#10494-1-AP), and HIF-1α (#20960-1-AP) were obtained from Proteintech (Wuhan, China), c-Fos (#4384), p-c-Fos (S32, #5348), c-Jun(#9165), p-c-Jun (S73, #9164), were purchased from Cell Signaling Technology, p-p38 (#AB4822) and IL-1R1 (#Ab106278) from Abcam USA, p-PI3K (#BS4605), PI3K (#AP0230), Akt (#BS1810), p-Akt (T308, #AA331) was obtained from Bioworld Technology (Nanjing, China), VE-cadherin (#E-AB-12772) from Elabscience. HRP-conjugated goat anti-rabbit secondary antibody was purchased from ZSGB-BIO (Beijing, China).