Anti nrp1 antibody

Human and mouse liver samples or cells were harvested and washed twice with phosphate-buffered saline (PBS) and then lysed with 1× sodium dodecyl sulphate (SDS) loading buffer. Equal sample quantities were electrophoresed on SDS–PAGE gels and transferred onto PVDF membranes. The membranes were blocked for 1 h with 5% (w/v) skimmed milk, incubated with the primary antibodies overnight at 4 °C, and with the secondary antibodies for 1 h at room temperature. The following primary antibodies were included: anti-NRP-1 antibody (1:1000), anti-VEGFR2 antibodies (1:100), anti-CD31 antibodies (1:500), anti-α-SMA antibodies (1:500, antibody acquired from Abcam); anti-Akt/pAkt antibody (1:1000), anti-ERK/pERK antibody (1:1000), anti-PI3K/pPI3K antibody (1:1000), anti-PLCγ-1/pPLCγ-1 antibody (1:1000), and anti-FAK/pFAK antibody (1:1000, acquired from CST). Anti-β-actin antibody (1:1000), anti-GAPDH antibody (1:1000) or anti-β-tubulin antibody (1:1000, acquired from CST) were used as an internal control in different experiments, respectively. Western blot results were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). β-actin, GAPDH or β-tubulin in the same membrane were used as internal controls. All the antibody bands were normalized to their expression.