Figure 3.

Effect of pre-treatment of papain on the phosphorylation status of key-regulatory proteins. HUVEC were seeded into fibronectin coated 25 cm² tissue culture flasks and cultured in EGM. After 24 hours medium was changed to EBM containing 10 μg/mL Papain and cells were incubated for further four hours. Cells were washed with PBS to remove papain and treated for 15 minutes with EBM containing 10 ng/mL VEGF. Untreated cells served as control. Protein was isolated and phosphorylation was measured using a multipathway ELISA kit. Data are shown as mean ± standard error of measurement with the numbers indicating p-values, black bars indicating VEGF treatment alone and open bars indicating pre-treatment with papain. Pretreatment with papain profoundly interferes with the ability of cells to respond to angiogenic stimuli. Phosphorylation of MEK1 and p38-MAPK were significantly downregulated, whereas ERK1/2 was significantly upregulated. Phosphorylation of Akt1 and SAPK/JNK was also downregulated but with p-values between 0.1 and 0.15.