Rabbit anti vegfa

Six rats per group were used for western blot detection at the corresponding sampling time. Before protein cleavage, PMSF (Sangon Biotech) was added to RIPA Lysis Buffer I (Sangon Biotech), lysed on ice for 1 h, and the supernatant was collected. After that, the protein concentration was measured by the BCA method. Absorb 50 g of protein per sample's concentration, mix with loading buffer in a 4:1 ratio, and dilute to 20 L. Fill it up with ddH2O. The concentrated gel and separation gel electrophoresis voltages are 80 V and 100 V. Electrophoresis is complete when bromophenol blue reaches the bottom of the gel. The PVDF membrane was blocked in 5% skim milk for 2 h after the protein sample was transferred. After blocking, the membrane was gently washed with TBST, rabbit anti‐VEGFA (1:100, Santa Cruz Biotechnology), mouse anti‐Notch1 (1:100, Santa Cruz Biotechnology), and rabbit anti‐Hes1 (1:100, Santa Cruz Biotechnology). Antibodies were incubated overnight at 4°C. After 16 h, the membrane was taken out and washed on the TBST shaker for 3−5 min. After washing, add anti‐HRP goat anti‐mouse IgG‐Fc II (Cell Signaling Technology) and shake for 1 h. Darkroom imaging with ECL color developing solution. ImageJ software was used to measure the gray value of VEGFA, Notch1, Hes1, and ‐actin proteins. Finally, the final relative expression was compared according to the ratio.

Proteins were isolated from snap frozen liver tissue by homogenising in ice-cold Tris-HCl (50 mM, pH 7.6) with protease inhibitor cocktail (Sigma-Aldrich Company Ltd., Gillingham, UK), PMSF, and EDTA. The protein concentration was determined using Bradford reagent. Cultured cells were lysed in ice-cold RIPA buffer. The protein concentration was measured using a BCA assay kit (Life Technologies Ltd., Paisley, UK). Equal amounts of protein were electrophoresed through 4–12% NuPAGE Bis-Tris Gels and transferred to PVDF membranes (Life Technologies Ltd., Paisley, UK). Blots were probed with the following primary antibodies: rabbit anti-DDAH1 (Abcam, Cambridge, UK); mouse anti-eNOS (BD, Oxford, UK), mouse anti-NF-κB, (Cell Signalling Technology, Danvers, MA, USA), mouse anti-4HNE (Abcam, Cambridge, UK), rabbit anti-Vegf-A (Santa Cruz Biotechnology, Inc, Dallas, TX, USA), mouse anti-Gapdh (loading control; Abcam, Cambridge, UK), and mouse anti-α-tubulin (loading control; Millipore, Watford, UK). Immune complexes were detected using HRP-conjugated secondary antibodies (Cell Signalling Technology, Danvers, MA, USA) and enhanced chemiluminescence (ECL) reagents (GE Healthcare Lifesciences, Little Chalfont, UK). Densitometric quantification was performed using Image J (US NIH, Bethesda, MD, USA; http://imagej.nih.gov/ij/).