2.7. Analysis of VEGFR2 signalling

Cancer cells were seeded in 6-well plates overnight, serum starved for 24 h, and then treated with or without rhVEGF for the indicated times. For some experiments, the cells were treated with DMSO or sunitinib (10 μM) for 1 h before addition of rhVEGF. After incubation, the cells were rinsed twice with PBS and lysed in M-PER buffer (Thermo Scientific, USA) containing protease and phosphatase inhibitor cocktails (Thermo Scientific). Protein in the lysates was quantified using a BCA Protein Assay Reagent Kit (Thermo Scientific). Samples were then analysed by western blotting (see below). The following primary antibodies were purchased from Cell Signaling Technology (USA): VEGFR2 (#9698, RRID: AB_11178792), phosphorylated (p)-VEGFR2 (Tyr1175) (#2478, RRID: AB_331377), ERK1/2 (#4695, RRID: AB_390779), p-ERK1/2 (#4370, RRID: AB_2315112), AKT (#4685, RRID: AB_2225340), p-AKT (#4060, RRID: AB_2341228), p-FAK (#8556, RRID: AB_10891442), p-P38 (#4511, RRID: AB_2139682), p-PLCγ (#8713, RRID: AB_10890863), p-SRC (#6943, RRID: AB_10013641), and β-actin (#3700, RRID: AB_2242334). Primary VEGFR2 antibody from Abcam (ab39256, RRID: AB_883437) was also used. A primary anti-OVA66 antibody was obtained from Santa Cruz Biotechnology (USA; #sc-271650, RRID: AB_10708575).