Detection and localization of P-Y-Stat5 and P-S-Akt in isolated neoplastic mast cells. (A) Expression of P-Y-Stat5 and tryptase was also evaluated by immunocytochemistry in isolated neoplastic MCs (from a patient with mast cell leukemia [MCL]) and normal isolated BM cells. (B) Detection of P-Y-Stat5 and P-S-Akt in neoplastic MCs obtained from patients with MCL in the absence or presence of a blocking phospho-peptide. (C) Immunodetection of P-Y-Stat5, P-S-Akt, and tryptase in the human HMC-1 neoplastic MC line. The P-S-Akt staining was also performed in the presence of a blocking phospho-peptide. (D,E) Cytoplasmic and nuclear extracts (CE and NE) were prepared from HMC-1 cells and analyzed by Western blotting using the indicated antibodies. (F) Detection of cytoplasmic P-Y-Stat5 in HMC-1 cells by FACS. Cells were cultured in control medium (solid line, open graph) or 1 μM PKC412 (solid line, gray graph) at 37°C for 4 hours followed by FACS using an antibody against P-Y-Stat5. The dotted line represents the buffer control. The isotype control exhibited a slight shift compared with the buffer control, but did not change after exposure to PKC412 (not shown). Representatives of 3 independent experiments are shown.