Quantitative Protein Analysis in Cell Signaling

Protein extraction and western blot analysis were carried out as previously described [26 (link),27 (link),28 (link)]. The blots were then incubated overnight at 4 °C with the primary antibodies against vascular endothelial growth factor receptor 1 (VEGFR1, also known as FLT1); VEGFR2, SRC–proto-oncogene non-receptor tyrosine kinase (SRC); protein tyrosine kinase 2 (PTK2, also known as FAK1); phospholipase C gamma 1 (PLCG1); KRAS GTPase proto-oncogene (KRAS); mitogen-activated protein kinase 3/1 (MAPK3/1, also known as ERK1/2) (Cell Signaling Technology, Danvers, MA, USA); phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA, also known as PI3K); B-Raf proto-oncogene, serine/threonine kinase (BRAF); small heat shock protein family B member 1 (HSPB1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and VEGFA (Novus Biologicals, Littleton, CO, USA).