Ab11939

Tumor tissues were ground in liquid nitrogen and treated with RIPA for 15∼30 min. Then the tissues or cells were ultrasonicated at 5 s × 4 times and centrifuged at 4°C and 10 000×g for 15 min. The supernatant was stored at −20°C. The protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membrane. After block by 5% skim milk for 2 h, the membrane was incubated in primary antibody (1:1000) at 4°C overnight. Next, the membrane was further incubated in secondary antibody (1:1000) at room temperature for 30 min. After washing with phosphate-buffered saline/Tween, the membrane was mixed with a chemiluminescent agent for 1 min and developed. Protein image processing system software and Quantity One software were adopted for scanning and calculation. Antibody: anti-p-VEGFR2 (ab38473, Abcam, USA); anti- VEGFR2 (ab11939, Abcam, USA); anti-PI3K (12402S, Cell Signaling, USA); anti-p-AKT (4060S, Cell Signaling, USA); anti-AKT(9272S, Cell Signaling, USA); anti-Cyclin B1 (ab181593, Abcam, USA); anti-Cyclin A(ab185619, Abcam, USA); anti-CDK1(ab32094, Abcam, USA); anti-Bax (ab32053, Abcam, USA); anti-Bcl-2(ab182858, Abcam, USA); and anti-GADPH(ab9485, Abcam, USA).

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.

Western blot analysis was carried out to analyze the expression of VEGFA (vascular endothelial growth factor A) and VEGFR2 (vascular endothelial growth factor receptor 2) in spleen, liver, BM, and skin. In brief, the total protein of the petrous part of the temporal bone samples was extracted using Radio-Immunoprecipitation Assay (RIPA) protein extraction reagent (Beyotime, Shanghai, China) supplemented with phenylmethylsulfonyl fluoride (Beyotime) at 4°C for 20 min. Samples were then centrifuged at 4°C (15 min, 14,000 × g). The protein concentrations were measured using a bicinchoninic acid (BCA) protein detection kit (Thermo, USA). The lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane. After blocking with 5% bovine serum albumin, the membrane was incubated with primary antibodies raised against VEGFA (Cat. No. ab46154; Abcam; 1:1000), VEGFR2 (Cat. No. ab11939; Abcam; 1:500), and GAPDH (loading control; ZSGB-BIO, Beijing, China). Goat antirabbit antibodies conjugated to horseradish peroxidase were used as secondary antibodies (1:500; SA00001-2, Proteintech, Rosemont, IL, USA), and proteins were visualized using enhanced chemiluminescence (Tanon, Tanon-5200, Shanghai, China). The band intensity was quantified using ImageJ software. Three independent experiments were performed.