Vegfa

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).

The rat livers and AML12 cells were lysed by RIPA buffer, separated by 10% polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membrane. After blocking in 5% Bovine Serum Albumin (BSA), the membrane was incubated with the phospho-JAK2 (Tyr931) (1:1,000, affinity), total JAK2 (1:1,000, proteintech), phospho-STAT3 (Tyr705) (1:1,500, abcam), total STAT3 (1:2000, abcam), IL-6 (1:500, bioworld), IL-18 (1:2000, bioworld), IL-1β (1:500, sino biological), TNFα (1:1,000, sino biological), HIF-1a (1:1,000, Novusbio), HIF-2a (1:1,000, Cell Signaling Technology), VEGFa (1:1,000, Novusbio) primary antibody seperately overnight at 4°C, and then was incubated with HRP conjugated secondary antibody for 1 h at room temperature. Western blots were displayed by advanced enhanced chemiluminescence kit and then semi-quantified by Clinx (Shanghai Qinxiang Scientific Instrument Co., Ltd).

VEGFA and β‐actin and Tubulin antibodies were purchased from Novus Biologicals and Cell Signaling, respectively. The protein lysates for all cells were separated using 12% SDS PAGE. Later, they were transferred onto 0.22‐μm PVDF membranes (Millipore) and then incubated with VEGFA antibody. Anti β‐actin served as control. Proteins were detected with Super Signal Chemiluminescence Substrate (Pierce, Thermo Scientific).