Anti vegfa

Proteins of specimens from three mice in each group were extracted by RIPA buffer (Sigma-Aldrich, United States) followed by high-speed centrifugation (12,000 g/min at 4°C for 10 min) at day 7. The protein concentration was determined using a BCA protein quantitative kit (Beyotime, China). 20 μg of total protein was separated by SDS-PAGE, electrophoresed, and transferred onto a PVDF membrane (Sigma-Aldrich, United States). The membrane was blocked with 5% BSA/TBST for 1 h, then incubated with the following primary antibodies: anti-CGRP (1:1,000, Santa Cruz, United States), anti-VEGFA (1:1,000, Abcam, United States) and anti-GAPDH (1:5,000, Signalway Antibody, United States) at 4°C overnight. The membrane was washed with TBST thrice and then incubated with HRP labeled secondary antibody (1:5,000, Abclonal, China) for 2-3 h at room temperature. The membrance was treated with the ECL blotting reagents (Zenbio, China). The protein bands were visualized with QuantityOne software (Bio-Rad, United States). Before incubating other primary antibody, the blots were rinsed in distilled water for 5 min, then soaked in the antibody stripping buffer (Servicebio, China) for 60 min to remove ECL substrates.

Whole-cell lysates for western blot analysis were prepared using standard radioimmunoprecipitation assay buffer. In brief, 30 μg protein were separated on 10% acrylamide/bis-acrylamide gels before transfer to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA) using CAPS buffer (10 mM 3-[cyclohexylamino]-1-propane sulfonic acid, pH 11; 10% methanol). The following primary antibodies were diluted in 5% w/v BSA (Sigma) in TBS-T: 1:1000 anti-Bcl-2 (Dako), 1:1000 anti-cleaved caspase 3 (Cell Signaling Technologies, Danvers, MA, USA), 1:30 000 anti-tubulin (Promega, Madison, WI, USA), 1:1000 anti-VEGFA (vascular endothelial growth factor, Abcam) and 1:1000 anti-NMYC (Abcam). Horseradish peroxidase-labeled secondary antibodies were used (Dako) and detection was carried out with Lumi-Light POD-substrate (Roche, Basel, Switzerland). Quantification of band intensities was performed using Image Lab Software 5.2.1 (Bio-Rad) and adapted to the corresponding loading control.

Total protein was extracted by RIPA according to the manufacturers’ protocol, containing protease inhibitors. Equal amounts of protein were separated by 10% SDS-PAGE gel and transferred to PVDF membranes under certain conditions. The primary antibody was then incubated overnight under 4 °C, the next day, the secondary antibody was incubated for 1–2 h at room temperature, followed by exposure to developer and storage of the images through Tanon 5200 (Tanon, China). The primary antibodies used above include anti-VEGFA (1:1000, abcam, USA) and anti-GAPDH (1:2000, Servicebio, Wuhan, China). Among them, GAPDH was used as an internal reference.

After treatment with siRNA, cells were collected, extracted with RIPA buffer (Beyotime, China), and boiled at 100 °C for 10 min. Then, the proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10%, Bio-Rad, China) and transferred to PVDF membranes (0.22 μm, Millipore). After blocking with 5% BSA, the membrane was incubated with the primary antibody and then secondary antibody. Equivalent protein loading was confirmed using anti-GAPDH (#2118, Cell Signaling Technology, United States). Anti-CERS1 (#ab98062, Abcam, United States), anti-GDF1(#bs-1794R, Bioss, China) anti-ATF4 (#11815, Cell Signaling Technology, United States), anti-VEGFA (#ab46154, Abcam, United States), anti-BIP (#ER40402, Huabio, China), anti-CHOP (#5554, Cell Signaling Technology, United States), anti-BAX (#5023, Cell Signaling Technology, United States), and anti-BCL2 (#4223, Cell Signaling Technology, United States) were used. A chemifluorescence kit (Bio-Rad, China) and imaging system (Bio-Rad, China) were used for visualization of blots. ImageQuant 5.2 (GE Healthcare) was used for quantification.