Ab17943

The proteins of tumor tissues were performed by building and construction authority (BCA) kit (Pierce Company). Samples from each group were isolated by SDS-PAGE electrophoresis (Mini-Protean-3, bio-Rad, Hercules, CA, USA) and then transferred to PVDF membrane (Millipore, Massachusetts, USA). Skim milk of 5% was used for sealing in 1 h. After washing with PBS, membranes were incubated with antibodies: anti-VEGF antibody (1:1,500, AF-493-NA, R&D Systems), anti-VEGFR2 antibody (1:1,000, ab11939, Abcam), anti-CD31 antibody (1^:800; AF3628, R&D Systems), anti-NF-kB p-p65 antibody (1^:1,000, ab86299, Abcam), anti-NF-kB p65 antibody (1^:1,000, ab19870, Abcam), anti-p-inhibitor of NF-κB (IκB)α antibody (1:2,000, ab19870, Abcam), anti-inhibitor of NF-κB (IκB)α antibody (1^:2,000, Abcam), anti-p-IκB kinase (IKK) antibody (1^:2,000, ab17943, Abcam), anti IKK antibody (1:2,000, ab17943, Abcam) and anti-β-actin (1^:1,000, ab179467, Abcam) overnight at 4 °C. The membranes were washed with PBS and incubated with IgG-HRP secondary antibody (1:800, ab205719, Abcam) at room temperature for 1 h. Signals detection was performed using ECL luminescent substrate (Thermo Fisher Scientific, Inc., Shanghai, China). Relative expression levels of each protein were normalized to β-actin.