Analysis of PKC activity in B-CLL cells.

(A) Membrane-associated PKC activity in particulate fraction. B-CLL cells were incubated for 10 minutes in the presence of 10 nM TPA for 20 minutes in the presence of 10 ng/mL IL-4 or in medium alone. After that time, cells were harvested and lysed, and the distribution of the enzyme between cytosolic fraction and particulate fraction was evaluated. PKC activity analysis was performed as described in “Patients, materials, and methods.” The cell extract was preincubated with 1 μM BisI for 20 minutes at room temperature before reaction was initiated. Values are mean ± SD for 6 different patients. Results are shown as the percentage of PKC activity present in the particulate fraction with respect to total PKC activity (cytosolic fraction plus particulate fraction). (B) Phosphorylation of PKC substrates in intact B-CLL cells. B-CLL cells were labeled with³²P as described in “Patients, materials, and methods,” in the absence or the presence of 5 μM BisI. Then cells were incubated with 10 nM TPA or 10 ng/mL IL-4 for 10 and 20 minutes, respectively. The figure shows the results corresponding to 1 representative patient from 4 patients analyzed who had similar results.