Figure 6

Saposin C activation of MAPK pathway is pertussis toxin-sensitive and PI3K/Akt-dependent. Cells were cultured up to 60% confluency in their maintenance media, washed with PBS, serum-deprived for 20 h, and then fresh basal culture media was added for an additional 4 h in the presence or absence of LY294002 (50 μM, 3 h), Wortmannin (10 μM, 15 min), U0126 (10 μM, 1.5 h), or pertussis toxin (200 ng/ml, 4 h). After pretreatment, saposin C (0.1 nM) was added directly to the cells and incubated for 5 min at 37°C. Cell lysates were prepared and 10 μg protein per sample was subjected to SDS-PAGE and immunoblotting using phospho-specific p42/44 MAPK antibody. For control loading, membranes were also probed or reprobed with p42/44 antibody to detect total p42/44 MAPK. Parallel tissue culture plates, treated in the same manner, were also tested for cell viability by trypan blue dye-exclusion assay. FBS at 10% final concentration was used as a positive control. Each experiment was performed in duplicate, and the assays were repeated three times.