4.10. Western Blot Analysis of VEGFR2 Phosphorylation

MS1 cells (250,000 cells/well) were seeded in 6-well plates in DMEM medium containing 10% FBS and 1% penicillin/streptomycin and allowed to adhere overnight. The next day, the cells were rinsed with DMEM containing 0.1% FBS and serum-starved overnight in the same medium. The day of the assay, 50 nM VEGF-A (PEPROTECH^®, Cranbury, NJ, USA) or 1 µM nanobodies were added to the cells in the starvation medium and incubated for 15 min at 37 °C. Afterwards, cells were immediately washed twice with ice-cold PBS and total cell lysates were prepared with scraping cells in 100 µL of 1× Laemmli sample buffer and boiled for 10 min at 100 °C. Proteins were separated on 8% SDS-PAGE and blotted onto PVDF membrane (Roche, Mannheim, Germany). The blots were then blocked with 2% BSA in TBS-T (0.05% Tween-20 in 20 mM Tris-buffered saline pH 7.2, TBS-T) buffer for 1 h at RT, followed by staining overnight at 4 °C for phosphorylated receptor using a rabbit monoclonal anti-VEGFR2 phospho-tyrosine 1175 antibody (19A10, Cell Signaling) diluted in 2% BSA in TBS-T. As a loading control, the lower part of the blot was stained for actin overnight with a mouse monoclonal anti-actin antibody (ICN Biomedicals, Irvine, CA, USA). Afterwards, the blots were incubated with IRDye800CW goat anti-rabbit secondary antibody (LI-COR) and IRDye800CW donkey anti-mouse secondary antibody (LI-COR) for 1 h at RT. Bound antibody was visualized with an Odyssey infrared scanner at 800 nm. Afterwards, blots were stripped using 1% SDS and 25 mM glycine pH 2, blocked with 2% BSA and then incubated with rabbit anti-VEGFR2 monoclonal antibody (55B11, Cell Signaling) overnight at 4 °C. After incubation with IRDye800CW goat anti-rabbit secondary antibody, the total VEGFR2 was visualized at 800 nm.