Figure 4

Activity of intracellular kinases in primary RCC cells of different metastatic potential after calcium treatment. (A) Activity of AKT, p38α, JNK, paxillin and PLCγ-1 was analyzed by measuring the phosphorylation status in primary RCC cells of patients developing no metastases or bone metastases within 5 years after nephrectomy via a human phospho-kinase array. Values of calcium treated cells (5 mM, 30 min) with or without pretreatment with NPS 2143 were related to untreated cells. The activity of AKT, PLCγ-1, p38α, JNK and paxillin was increased in bone metastasizing cells after treatment with calcium. This effect was inhibited using NPS 2143. The columns represent the amount of antigen determined by computer-aided integration of the bands after subtraction of the background. Demonstrated are the mean values of each lysate in% of untreated non-metastasizing cells (= without metastases). (B) Western blot analysis of phosphorylated AKT and ERK in cell lysates of untreated, calcium treated (5 mM, 30 min) or NPS 2143 (5 μM, 30 min) following by NPS 2143 + calcium (30 min) treated cells, 100 μg per lane. β-actin served as an internal control.