Western Blot Analysis of Signaling Pathways

Unstimulated or stimulated endothelial cells, and serum (10%) stimulated tumor cells were treated with 2-DG or other agents, incubated for different time periods and lysed. Protein concentration was determined from lysates by the BCA assay (Thermo Scientific, Rockford IL). 10-20 μg proteins were prepared with 4X Laemmeli sample buffer and separated in 10% or 4-20% Mini-Protean TGX gel (BioRad, Richmond, CA), transferred to 0.45 μm pore sized PVDF membranes (Bio-Rad) and probed (1:1000 dilution) for Akt pSer⁴⁷³, S6 pSer^240/244, Erk pThr²⁰²/Tyr²⁰⁴, GSK-3β pSer⁹, PERK pThr⁹⁸⁰, cleaved-Caspase-3, cleaved-PARP, total-VEGFR2, VEGFR2 pTyr¹¹⁷⁵, and PLC-γ1 pTyr⁷⁸³ (Cell signaling, Danvers, MA), GSK-3β pTyr²¹⁶ (Calbiochem), and GAPDH (1:20,000 dilution) for loading control (Rockland, Gilbertsville, PA). Following probing, membranes were processed as previously described (15 (link), 18 (link)). Band intensities of replicate Western blot figures were quantified with ImageJ software, normalized to the corresponding GAPDH bands, and results presented as percentage of control (untreated cells) +/− SEM.