Comparison of flow cytometric quantification of specifically phosphorylated signaling intermediates (phospho-flow) to Western blotting. (A) HEL cells were treated with different concentrations of the JAK2-selective agent TG101209 for 4 hours. Aliquots were taken from each concentration and divided in half, with one half analyzed by Western blotting (top panel) and the other half by phospho-flow analysis (bottom panel). The percent inhibition by each method is shown. Inhibition of phospho-STAT5, phospho-STAT3, and phospho-ERK was demonstrated using both methods. (B) To ensure that the phospho-flow assay was robust and reproducible in patient samples, we performed repeat experiments, assessing 4 signaling pathways (phospho-ERK, phospho-STAT5, phospho-STAT3, and phospho-AKT) in each experiment for 5 patients. For each patient, experiments were performed in triplicate, each at a different time point, from frozen aliquots of cells. A high degree of correlation can be seen between runs for each individual patient.