Affinity purified secondary antibodies conjugated with horseradish peroxidase

Cells were lysed in 1% Nonidet P-40 (NP-40) lysis buffer (50 mM Tris-HCL, PH 7.4, 10% glycerol, 200 mM NaCl and 2 mM MgCl₂) containing protease inhibitors. Protein lysates were loaded onto 8–15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Membranes were blocked with 5% non-fat milk in 1× TBS containing 0.3% Tween-20, then incubated with the following primary antibodies overnight at 4°C: rabbit anti-human VEGFR2 (SC-504), c-Jun (SC-1694); mouse anti-human VE-cadherin (SC-9989), CD31 (SC-365804), CD146 (SC-18837), MDM2 (SC-965), p53 (SC-126), phosphor-c-Jun (SC-822), phosphor-JNK (SC-6254), JNK (SC-1648), mouse anti-human β-actin conjugated with HRP (SC-47778HRP) (Santa Cruz Biotechnology; Santa Cruz, CA, USA); mouse anti-GAPDH (MAB374; Millipore Sigma); rabbit anti-human Tie-2 (7403), Bmi-1(6964), PDGFR-a (5241), PDGFR-β (4564), p21 (2947) (Cell Signaling; Danvers, MA, USA); mouse anti-human SMA-alpha; CBL171; Millipore Sigma). Affinity-purified secondary antibodies conjugated with horseradish peroxidase (Jackson Laboratories; West Grove, PA, USA) were used and immunoreactive proteins were visualized by SuperSignal West Pico chemiluminescent substrate (cat:34578; Thermo Scientific; Rockford, IL, USA). Here, and thereafter, in vitro experiments were performed three independent times to verify the reproducibility of the data.

Whole cell lysates were prepared with 1% Nonidet P-40 (NP-40) lysis buffer (50 mM Tris-HCL, PH 7.4, 10% glycerol, 200 mM NaCl and 2 mM MgCl2) containing protease inhibitors. Protein samples were loaded onto 8%–15% SDS-PAGE. Membranes were blocked with 5% non-fat milk in 1X TBS wash buffer containing 0.3% Tween-20, then incubated with the following primary antibodies overnight at 4°C: rabbit anti-human VEGFR2, mouse anti-human CD31 (Santa Cruz Biotechnology, Santa Cruz, CA, United States); rabbit anti-human Bmi-1, PDGFR-α, phosphor-PDGFR-β, PDGFR-β, phosphor-Tie-2, Tie-2, phosphor-AKT, AKT (Cell Signaling, Danvers, MA, United States); mouse anti-human smooth muscle actin-α (SMA-α), mouse anti-GAPDH (Millipore/Sigma). Affinity-purified secondary antibodies conjugated with horseradish peroxidase (Jackson Laboratories, West Grove, PA, United States) were used and immunoreactive proteins were visualized by SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL, United States).