The kinase-phosphatase dynamic equilibrium determines the activity profile of signaling intermediates. Panel A, shows the results of a pulse-chase experiment (see text) where the indicated intermediates were immunoprecipitated and their phosphorylation status monitored either by Western blot (with phospho-specific antibodies, lane 1), or by autoradiography (lane 2). Lane 3 shows the results of a parallel experiment where phosphatase inhibitor (Sodium vanadate) was also included during the chase period. Lane 4 compares the amount of the parent protein present in each group as detected by Western blot using antibodies specific for the individual proteins. Panel B provides a graphical depiction of the relative stability of the phosphorylated forms of each of these intermediates after they reach the peak, when detected either by Western blot (left hand side), or by autoradiography (middle). The profile obtained in parallel groups that included phosphatase inhibitor is also shown (right hand side). Intensity values obtained for the data shown in panel A are plotted here, and values are the mean (± S.D.) of two independent experiments. The rate of turnover of the 32P-labelled γ phosphate of ATP, in these cells, is shown in panel C. Panel D shows the results of co-immunoprecipitation experiments where the indicated signaling intermediates (indicated on the left of the blots) were immunoprecipated from cells stimulated for various times as shown. These immunoprecipitates were then examined by Western blot analysis for the association of various cellular phosphatases. The results obtained are shown here and the identified phosphatases are indicated on the right of the blots.