Rabbit anti myb antibody

Lentivirus was commercially obtained and titration was performed according to the directions of the manufacturer (Genechem, China). siRNA-mediated knockdown of MYB (Gene ID: 498982) in BMVECs was achieved by transfection of lentivirus using a siRNA oligonucleotide kit according to the kit instruction. A commercially procured non-targeting siRNA from the same corporation was used as a negative control. The incorporation of siRNAs into BMVECs was detected by fluorescence microscopy. Western blot assay was used to identify the expression of MYB in siRNA-transfected BMVECs by employing the rabbit anti-MYB antibody (Abcam, 1:200) while quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to test the levels of mRNA.

Cells were harvested and lysed in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Non-idet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM phenylmethylsulfonyl fluoride). A Bio-Rad protein assay kit was used to determine protein concentrations, with bovine serum albumin acting as a reference. Samples containing 30 μg of protein were put on 10% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Membranes were incubated with specific primary antibodies including rabbit anti-VEGF antibody (Abcam, 1:1,000), rabbit anti-MYB antibody (Abcam, 1:400) and rabbit anti-β-actin antibody (Abcam, 1:2,000), then membrans were incubated with horseradish peroxidase-conjugated secondary antibody (Sigma, 1:10,000). Proteins were evaluated using a SuperSignal West Pico chemiluminescence kit (Thermo Scientific, United States).