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. 2013 Feb 11;288(13):8838–8848. doi: 10.1074/jbc.M112.437186

FIGURE 2.

FIGURE 2.

Inhibition or depletion of PP1 inhibits CaMKII dephosphorylation and apoptosis. A, depletion of PP1 and PP2A inhibits recombinant CaMKIIα Thr-286 dephosphorylation. Upper panel, GST-CaMKIIα (TT305/6AA) was phosphorylated in egg extract in the presence of G6P as in Fig. 1A. Beads were collected, washed, and then incubated in egg extract depleted with Sepharose or microcystin-Sepharose (MC-LR). At the indicated times, samples were taken and analyzed by SDS-PAGE, Coomassie Blue staining, and autoradiography. Lower panel, depletion of PP1 and PP2A was confirmed by immunoblotting for PP1β, PP2A, and GAPDH as a loading control. n = > 3 independent experiments. Seph, Sepharose. B, depletion of PP1 and PP2A inhibits endogenous CaMKII Thr-286/287 dephosphorylation. Upper panel, egg extracts were depleted as in A. Samples were taken at the indicated times and immunoblotted for CaMKIIα and pCaMKII Thr-286/287. Lower panel, depletion of PP1 and PP2A was confirmed by immunoblotting for PP1β, PP2A, and GAPDH as a loading control. n = 3 independent experiments. C, depletion of PP1 and PP2A inhibits apoptosis. At the indicated times, samples were taken from egg extract depleted with Sepharose or MC-LR and analyzed for caspase 3/7 (C3/7) activation using a caspase 3/7 GLO assay (Promega). n = > 3 independent experiments. D, inhibition of PP1 and PP2A with okadaic acid inhibits recombinant CaMKIIα Thr-286 dephosphorylation. Left panel, GST-CaMKIIα (TT305/6AA) was phosphorylated in egg extract in the presence of G6P as in Fig. 1A. Beads were collected, washed, and then incubated in a fresh aliquot of egg extracts containing 1 nm or 10 μm okadaic acid. Samples were taken at the indicated times and analyzed as A. Right panel, the same experiment as displayed in the left panel, conducted independently. n = > 3 independent experiments. E, inhibition of PP1 and PP2A with okadaic acid inhibits endogenous CaMKII Thr-286/287 dephosphorylation. Egg extracts were incubated at room temperature in the presence of 10 μm okadaic acid. Samples were taken at the indicated times and immunoblotted for CaMKIIα and pCaMKII Thr-286/287. n = 2 independent experiments. F, inhibition of PP1 and PP2A with okadaic acid inhibits apoptosis. Egg extracts were incubated in the absence or presence of okadaic acid (1 nm or 10 μm), and samples were taken at the indicated times and analyzed for C3/7 activation using a caspase 3/7 GLO assay (Promega). n = 3 independent experiments.