Rabbit antihuman bmi 1

Protein lysates from UM-HMC cells and dissociated xenograft MEC tissues were prepared using 1% Nonidet P-40 (NP-40) lysis buffer and loaded onto 9–12% SDS-PAGE gels. Membranes were incubated overnight at 4 °C with 1:1000 dilution of the following antibodies: rabbit antihuman p-AKT Ser 473 (Cell Signaling, Danvers, MA, USA), mouse antihuman AKT (Cell Signaling), rabbit antihuman p-Rictor Thr1135 (Cell Signaling), mouse antihuman Rictor (Santa Cruz Biotechnology; Dallas, TX, USA), rabbit antihuman p-mTOR Ser 2448 (Santa Cruz), rabbit antihuman mTOR (Cell Signaling), mouse antihuman p-4E-BP1 Ser 65 (Santa Cruz), mouse antihuman p-4E-BP1 (Santa Cruz), rabbit antihuman p-S6K1 Thr 421/Ser 424 (Santa Cruz), rabbit antihuman S6K1 (Cell Signaling), rabbit antihuman Bmi-1 (Cell Signaling). As surrogate for loading control we used Western blot performed with 1:100,000 antihuman β-actin antibody (Santa Cruz). Membranes were exposed to 1:10,000 affinity-purified secondary antibodies conjugated with horse-radish peroxidase (Jackson Laboratories; West Grove, PA, USA). Immunoreactive proteins were visualized by SuperSignal West Pico chemiluminescent substrate (Thermo Scientific).

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).