Anti p fak

The HUVEC line EA.Hy926 was obtained from American Type Culture Collection (ATCC, Manassas, VA). HUVECs were cultured with complete culture medium (RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicilin–streptomycin, 1% glutamine) and maintained at 37°C with 5% CO₂. Trypsin-EDTA was used to passage cells. All above agents were obtained from Gibco (Grand Island, New York, USA).
DTX was purchased from Xieli Pharmaceutical (Chengdu, China). CCK-8 kit and Annexin V-FITC Apoptosis Detection kit were purchased from Dojindo Laboratories (Shanghai, China) and KeyGEN Biotechnology (Nanjing, China), respectively. Anti-proliferative cell nuclear antigen (anti-PCNA) and anti-p53 were obtained from Proteintech Group (USA). Anti-β-actin, anti-GAPDH, anti-PECAM-1 and anti-integrin β1 were purchased from Santa Cruz Biotechnology (California, USA). Anti-VE-cadherin, anti-FAK, anti-pFAK, anti-Rho, anti-ROCK, anti-MLC2 and anti-pMLC were purchased from Abcam (Cambridge, UK). Anti-p190 RhoGEF was purchased from Bioss (Beijing, China). Y-27632 was purchased from Selleck chemicals (USA). HRP-conjugated and FITC/PE-conjugated anti-mouse/rabbit/goat secondary antibodies were obtained from Biosynthesis Biotechnology (Beijing, China).

The sections of tumor tissues were dewaxed, rehydrated, quenched of endogenous peroxidase, blocked by 5% BSA, and incubated with the primary antibodies: anti-p-FAK, anti-p-ERK, anti-NF-κB (1:200, Abcam, Cambridge, UK) overnight at 4 °C, and followed by signal amplification using the ABC HRP Kit (Thermo, MA, USA) for 2 h at room temperature, and the nucleus was stained with DAPI. All immunofluorescence images were captured by FV1000 confocal microscope (Leica, Barnack, Germany) and the intensity of protein expression was calculated by image J software.