α akt

For lysates, cells were serum deprived for 2 h and stimulated or not with 30 ng/ml VEGF-A for 15 min. When used, inhibitors were added to cells for 1 h prior to growth factor stimulation. Total proteins were extracted in Laemmli buffer (2.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol), quantified and equal amounts of each sample were resolved by SDS-PAGE and transferred to PVDF membrane. After blocking with TBS/0.1% Tween 20/5% BSA, membranes were incubated with primary antibody overnight at 4° C. The following primary antibodies were used: rabbit α-pY1175 VEGFR2, α-VEGFR2, α-pS473 Akt, α-pT308 Akt, α-Akt, α-pS235/236 S6, α-pS65 and pT37/46 4EBP1 (all from Cell Signaling Technology), α-β actin (Santa Cruz Biotecnology), α-p110 α (ThermoFisher Scientific). Immunoreactive proteins were identified with secondary antibody coupled to HRP antibody and visualized by ECL. Quantification of the bands was done by NIH ImageJ on representative western blots; band intensities of phosphorylated Akt were normalized on total Akt, and analogously those for phosphorylated VEGFR2 were normalized on total VEGFR2, whereas band intensities of phosphorylated S6 and 4EBP1 were normalized on β actin.

Cell suspensions were washed in PBS, pelleted, incubated in RIPA lysis buffer, and immunoblotting was performed as described previously28 (link). The following antibodies were utilized: α-FAK C-20 (Santa Cruz, Dallas, TX; [0.2 μg/ml]), α-ERK p44/42 (Cell Signaling, Danvers, MA; [1:2000]), and α-AKT (Cell Signaling; [1:1000]).

The following reagents were used in cell culture experiments: cisplatin (Tocris, 2251), etoposide (Millipore Sigma, E1383), LY2874455 (Selleckchem, S7057), infigratinib (Selleckchem, S2183), erdafitinib (Selleckchem, 8401), cabozantinib malate (Selleckchem, S4001), and lucitanib (MedChemExpress, HY-15391). cisplatin was dissolved in normal saline for stock preparations. All other small molecules were dissolved in DMSO for stock preparations. The following antibodies were used for immunoblotting experiments: α-p-FRS2 antibody (Cell Signaling Technology, 3864 S), α-p-Erk1/2 (Cell Signaling Technology, 9101 S), α-Erk1/2 (Cell Signaling Technology, 9102 S), α-p-Akt (S473) (Cell Signaling Technology, 9271 S), α-Akt (Cell Signaling Technology, 9272 S), α-ETV4 (Abcam, ab189826), and α-ETV5 (Thermo Fisher, PA5-66555).

Cells were centrifuged, washed once with cold PBS and subsequently lysed in Nonidet-40 lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 1 mM PMSF and one tablet of Roche EDTA-free protease inhibitor cocktail (Sigma-Aldrich) per 50 mL) for 10 min on ice. Lysates were then centrifuged (13,000 rpm, 10 min, 4 °C) and proteins were quantified with the Bradford assay using γ-globin as a standard (Bio Rad). Cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes Protran BA85 (GE Healthcare). The membranes were incubated with α-SLC16A1 (Santa Cruz Biotechnology; sc-365501 mouse mAb 1:500 dilution), α-SLC16A3 (Santa Cruz Biotechnology; sc-376140 mouse mAb 1:500), α-FLAG (Sigma-Aldrich; F1804 mouse mAb 1:1000), α-HSP90 (BD Biosciences; 610418 purified mouse Ab 1:2000) or α-AKT (Cell Signaling Technologies; #4685 rabbit mAb 1:1000) and visualized with goat-anti-mouse IgG (115-035-003) or goat-anti-rabbit IgG (111-035-003) horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch; 1:5000) utilizing the ECL western blotting system (Thermo Fisher Scientific).