FIG. 6.

Blocking PKCα and PKCβ activities in L6 cells with antisense oligonucleotides or LY379196. L6hIR2 cells were preincubated with 50 nM LY379196 or transfected with either specific PKCα or PKCβ antisense oligonucleotides (ASPOα and ASPOβ, respectively). Oligonucleotides with the same base composition as the PKCα and -β antisense oligonucleotides but with random sequence (POα and POβ, respectively) were used for control. Cells were then exposed to 100 nM insulin for 30 min, solubilized, and precipitated with specific PKCα (middle) or PKCβ (bottom) antibodies. PKC activity in the immunoprecipitates was assayed as described in Materials and Methods and is plotted as radioactivity incorporated in the Ac-MBP(4-14) substrate. Bars represent the means ± standard deviations of duplicate determinations in three independent experiments. For control (top panel), lysates (300 μg of protein/assay) were subjected to SDS-PAGE and Western blotted with PKCα or PKCβ antibodies as indicated.