Figure 2.

(A) Whole cell lysates from indicated cell lines was used to determine the protein levels of RON using immunoblot analysis. β-actin was used as the loading control. Blot shown are representative of two individual experiments. (B). RON mRNA levels following 24 h 2-ME₂ treatment. Data presented is from 3 independent experiments. (C). Protein levels of RON following treatment with 2-ME₂ (24 h) in PC-3 and DU145 cell lines using immunoblot analysis. β-actin was used as the loading control. A representative blot from three individual experiments is shown (D). Impact of 2-ME₂ on RON kinase activity was determined using ADP-Glo^TM Kinase Assay (Promega Corporation, Madison, WI). The assay is based on monitoring the production of ADP concentration using luminescence which is directly proportional to kinase activity. Kinase reaction was performed essentially as per manufacturer’s protocol in the presence of increasing concentrations of 2-ME₂. To test if 2-ME₂ interferes with assay components, kinase reaction was performed excluding RON kinase and considered as background. Kinase activity is expressed as relative to kinase activity in the absence of 2-ME₂. Data presented is an average of triplicate measurements.