PP1 activity is involved in the dephosphorylation of BAD, and BAD
dephosphorylation during act-D-induced apoptosis is inhibited by
electroporation of Inh3(D49A). A, HL-60 cells were treated with 1
μm or 25 nm OA for 5 h, in the absence and presence
of act-D (4 μm), as indicated. Cell lysates were Western blotted
with a phosphospecific antibody for ERK (top), and with an antibody
against ERK (bottom). B, HL-60 cells were treated with 1
μm or 25 nm OA for 5 h. Cells were then analyzed for
BAD by Western blotting. The positions of phosphorylated (P-BAD) and
nonphosphorylated BAD are indicated. Data for A and B are
representative of three experiments. C, HL-60 cells were
electroporated with or without purified His-Inh3(D49A) prior to treatment with
act-D for 5 h or without act-D, as indicated. Cell lysates were Western
blotted for Inh3, BAD-phosphoserine 112, BAD, and PP1α. The data are
representative of three independent experiments.