Figure 3.

PKCε interacts with and phosphorylates VDAC in vitro. A, Equal amounts of recombinant GST or GST-PKCε fusion proteins conjugated to glutathione-Sepharose beads were incubated with mixes containing [³⁵S]-labeled VDAC1, ANT1, HKII, or CypD produced by in vitro translation (IVT). The complexes were pulled down and subjected to SDS-PAGE and either autoradiography (top 4 panels) or immunoblotting with anti-GST antibody (bottom panel). Lane 1 contains GST alone plus the respective in vitro translation product; lane 2 contains GST-PKCε plus the respective in vitro translation product; lane 3 is blank; and lane 4 contains the respective in vitro translation product as positive control (n=3). B, Recombinant GST-VDAC1 fusion protein was incubated with γ[³²P]-ATP plus increasing amounts of recombinant PKCε and subjected to SDS-PAGE and either autoradiography (top panel) or immunoblotting with anti-GST antibody (bottom panel). Lanes 1 through 4 contain radio-labeled GST-VDAC1 after incubation with 0, 10, 50, and 100 ng of recombinant PKCε, respectively (n=3). IB indicates immunoblotting.