Fig 5. Bone-derived OPN induces phosphorylation of WNK1 and PRAS40.

MDA-MB-231 human breast cancer cells were exposed to basal media, BMCM and BMCM depleted of OPN (BMCM ΔOPN) for 2 hours, and cell lysates were harvested. (A) Cell lysates were assessed using with Human Phospho-Kinase Array membranes (ARY003B, R&D Systems) overnight at 4°C. Biotinylated detection antibodies were applied and membranes were visualized using chemiluminescence. Densitometry analysis was performed using the Protein Array Analyzer for ImageJ (N = 3 for each media condition). * = significantly different than basal media; ϕ = significantly different than BMCM (P≤0.05). Only proteins with significantly different phosphorylation or expression between at least two treatments are shown. Proteins within the rectangular box demonstrated a similar pattern of phosphorylation to each other (increase in response to BMCM, with a subsequent decrease upon depletion of bone-derived OPN). (B) Phosphorylation patterns for PRAS40 and WNK1 were successfully validated using immunoblotting (N = 3).