Figure 4

ARC and sangivamycin inhibit PKC substrate phosphorylation, PKCδ kinase activity, RNA polymerase II phosphorylation and P-TEFb kinase activity. A) Representative blot of the effects of ARC, sangivamycin, toyocamycin, fludarabine and thioguanine on the endogenous PKC activity (upper panel) and TPA-stimulated PKC substrate phosphorylation (lower panel). Cells were incubated for 9 h with 100 μM drug. To stimulate PKC, 5 μM TPA was included during the last 2 h of incubation. Lysates were prepared and probed for PKC substrates containing phospho-serine. B) Activity of PKCδ to incorporate ³²P from [γ-³²P] ATP into PKC substrate peptide 2 in the presence of the indicated concentrations of drugs. C) Activity of the recombinant PKA catalytic subunit to phosphorylate the substrate Kemptide, in the presence of the indicated concentrations of drugs and the PKA inhibitor peptide. D) Upper panel, MCF7 cells were incubated with 100 μM ARC, sangivamycin, toyocamycin, fludarabine and thioguanine for 3 h before lysates were prepared and probed for phosphorylated RNA polymerase II. Lower panel, densitometric analysis of western blots. Data is presented as the total phosphorylated RNA polymerase as a propotion of actin band intensity. E) Purified P-TEFb was incubated with substrate (PDKtide) in the absence or presence of the indicated concentrations of drug. The effects of the drugs are presented as % of control.