Fig. (5) The human phospho-MAPK profile array and western blotting assays detects the phosphorylation of ERK1 in AG2034 resistant cells. 1.2 mg of cell lysate was used on each array from R & D systems (A, B and C). (A) Drug-naïve parent DU145 cells maintained in 0HPX media. (B) Drug-resistant DU145 cells cultured in 0HPX media continuously supplemented with 50 nM AG2034. The arrow points to duplicate spots corresponding to ERK1. (C) X-ray film images of the array signals from panels A and B were scanned and quantified using the imageJ free software by the NIH. Results are representative of three independent experiments. (D) Western blot analysis shows that AG2034 resistant DU-145 cells contain readily detectable levels of phosphorylated ERK1/2 as opposed to drug-naïve parental cells in which no evidence for their presence was found. Similar levels of total ERK1/2 proteins were found in both parental and drug-resistant cells.