TPO stimulation of HEL cells results in activation of PI 3-K. HEL cells were stimulated with 100 ng/mL TPO for 0 to 60 minutes at 37°C, lysed in PI 3-K lysis buffer, and then immunoprecipitated with anti-PY MoAb (A) or anti-p85 serum (B). Samples were then tested for PI 3-K activity as described in Materials and Methods. Data are expressed as means ± SD of five separate experiments. (C) HPLC analysis of deacylated (³²P) PIP produced by an anti-p85 immunoprecipitate of HEL cells stimulated by TPO for 15 minutes (○, left peak) or by a total HEL cell lysate used as a standard source of PI-4-P ( — , right peak). Equivalent counts of (³²P) PIP, obtained from an anti-p85 immunoprecipitate or total HEL cell lysates, were separated by thin-layer chromatography, deacyled, and subjected to HPLC separation, as described in Materials and Methods.