Anti phospho vegfr2

The antibodies including anti-HIF-1α, anti-VEGF, anti-CD31 (PECAM-1), anti-VHL, anti-WSB-1, anti-β-actin, anti-α-tubulin and fluorescein isothiocyanate (FITC)-coupled secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-VEGFR2, anti-phospho-VEGFR2, anti-AKT, anti-phospho-AKT, anti-mTOR, and anti-phospho-mTOR were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-labeled secondary antibody and goat anti-rabbit IgG-biotin secondary antibody were obtained from Abcam (Cambridge, MA, USA). The z-ligustilide, n-butylidenephthalide, and other chemical reagents used in this study were purchased from Sigma (St. Louis, MO, USA)

Total protein from the collected cell lines and tissue was extracted in PRO-PREP Protein Extraction Solution (Intron Biotechnology, Seongnam, Korea) on ice according to the manufacturer’s instructions. The protein concentration was measured using the Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA) as described previously [43 ]. The collected proteins (30 μg) were separated using sodium dodecyl sulphate gel electrophoresis, and the protein bands were transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h and incubated with antibodies against anti-cyclin-D1 (1:1000; LSBio, Seattle, WA, USA) and anti-phospho-VEGFR2 (1:1000; Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight. The membranes were incubated with anti-mouse or anti-rabbit immunoglobulin G (Santa Cruz Biotechnology, Dallas, Texas, USA) as the secondary antibodies (1:2000) for 1 h. Immunoreactive bands were normalised to β-actin (1:1000; Santa Cruz) or VEGFR2 (1:1000; Cell Signaling Technology) and visualised using SuperSignal West Pico PLUS Chemiluminescent substrate (Advansta, Menlo Park, CA, USA).

Human cervical carcinoma cell lines (CaSki and SiHa) and human embryonic kidney 293T (HEK293T) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in monolayer cultures according to ATCC recommendations. HUVECs were purchased from Clonetics (Walkersville, MD, USA), and seeded on 0.3% gelatin-coated dishes (Sigma, St. Louis, MO, USA) using the EGM-2 BulletKit medium (Clonetics). Wortmannin and LY294002, which are PI3K inhibitors, were obtained from Sigma (St. Louis, MO, USA). The following antibodies were used in this study: anti-HPV16 E6 (ab226447), anti-IgG (Abcam, Cambridge, UK), anti-IRF-1, anti-Flag, anti-cyclin D1, anti-CDK4, anti-NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Bax, anti-Bcl-xL, anti-Bcl-2, anti-p53, anti-VEGFR-1, anti-VEGFR-2, anti-phospho-VEGFR-2(Tyr-1175), anti-HIF-1α, anti-PI3K, anti-phospho-PI3K, anti-Akt, anti-phospho-Akt(Ser473), anti-PDK-1, anti-phospho-PDK-1(Ser241), anti-mTOR, anti-phospho-mTOR(Ser2448), anti-4E-BP1, anti-phospho-4E-BP1(Thr70) (Cell Signalling, Beverly, MA, USA), anti-p21, anti-VEGF (Ab-1; Oncogene, Cambridge, MA, USA), and anti-β-actin (Sigma, St. Louis, MO, USA).

Endothelial growth medium-2 (EGM-2) was obtained from Lonza (Walkersville, MD, USA). Minimum essential medium (MEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Grand Island, NY, USA). Recombinant human vascular endothelial growth factor (VEGF₁₆₅), recombinant human basic fibroblast growth factor (bFGF) and laminin were obtained from Koma Biotech (Seoul, Korea). Matrigel and Transwell chamber systems were obtained from BD Biosciences (San Jose, CA, USA) and Corning Costar (Acton, MA, USA), respectively. Anti-hypoxia inducible factor-1α (HIF-1α) antibody was purchased from BD Biosciences. Anti-phospho-VEGFR2, anti-VEGFR2, anti-phospho-Akt, anti-Akt, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-NFκB, anti-NFκB, anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-p38, anti-MMP-2, anti-MMP-9 and anti-β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

The tissue was homogenized in RIPA lysis buffer system with a protease inhibitor cocktail and phosphate inhibitors (Santa Cruz Biotechnology, Inc., CA). The protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific Corp., MA). The protein lysates were separated by SDS-PAGE and electroblotted onto PVDF membranes. The membranes were then blotted with primary antibodies; antiphospho-VEGFR2 (Cell Signaling Technology, MA; rabbit monoclonal, Cat # 2478; and polyclonal, Cat # 2471; both diluted 1:1,000), antiphospho-AKT (Cell Signaling Technology, MA; polyclonal, Cat #9271; diluted 1:1,000), anti-VEGFR2 (Cell Signaling Technology, MA; rabbit monoclonal, Cat #2479; diluted 1:1,000), anti-AKT (Cell Signaling Technology, MA; polyclonal, Cat #9272; diluted 1:1,000), and GAPDH (Cell Signaling Technology, MA; rabbit monoclonal, Cat #2118; diluted 1:1,000), followed by a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, MA; Cat #7074, diluted 1:1,000). Protein bands were visualized by chemiluminescence using a Bio-Rad ChemiDoc Imaging Systems with PicoEPD Western reagent kit (ELPIS BIOTECH, Inc., Daejeon, Korea).