Leptin effects on VEGF and VEGFR2 protein and mRNA in MCF-7 and MDA-MB231 cultures. Vascular endothelial growth factor (VEGF) protein and mRNA and vascular endothelial growth factor receptor type 2 (VEGFR2) protein and mRNA levels in MCF-7 breast cancer (BC) (a,c) and MDA-MB231 BC cells (b,d), respectively. The cells were cultured for 48 hours in medium containing leptin (0 to 6.25 nM, equivalent to 10 to 100 ng/ml) and/or pegylated leptin peptide receptor antagonist 2 (PEG-LPrA2) (3 μM). Levels of VEGF protein in the supernatants determined by ELISA. Levels of VEGFR2 protein in cell lysates determined by immunoprecipitation/western blot. β-Actin used as loading control for western blot determinations. VEGF and VEGFR2 mRNA determined by real-time RT-PCR and expressed as relative values to basal conditions. All data were derived from a minimum of three independent experiments using different cell preparations. *P < 0.05, comparing levels of basal cells with treated cells.