PKCδ is constitutively activated in B-CLL cells.

(A) A sample of 300 μg total protein content of 8 different freshly isolated B-CLL cells was immunoprecipitated with an antibody against PKCδ and in vitro kinase assay was performed in the absence (lanes 1-8) or presence (lane 9) of Rottlerin using histone H1 as an exogenous substrate. Proteins were analyzed by SDS-PAGE and a phosphorylated form of histone H1 was detected by autoradiography. (B) A sample of 100 μg total protein content of 7 different B-CLL cells was analyzed by immunoblot with an antiphosphothreonine 505 antibody specific for PKCδ. Samples from panel A were different from those shown in panel B. To investigate the influence of PI-3K activity on PKCδ, cells were incubated with 10 μM LY294002 and lysed after the indicated time period. Then PKCδ was immunoprecipitated with a specific antibody. (C) Tyrosine phosphorylation was detected by using an antiphosphotyrosine 4G10 antibody. (D) PKCδ kinase activity was measured by a in vitro kinase assay as described in (A).