Figure 3. PP1 binding is required for caspase-2 activation.
(A) Glutathione sepharose-bound GST-C2 pro WT, V18A/L20A, or V106A/H108A was incubated in extract, resolved by SDS-PAGE, and immunoblotted for PP1. NS = non-specific.
(B) Glutathione sepharose-bound GST-C2 pro WT, V18A/L20A, or V106A/H108A was incubated in buffer with recombinant PP1 catalytic domain. Sepharose-bound proteins were resolved by SDS-PAGE and immunoblotted for PP1. NS = non-specific.
(C) Glutathione sepharose-bound GST, GST-C2 pro WT, or V106A/H108A was prephosphorylated with [γ-32P]ATP. Samples were resolved by SDS-PAGE and examined by autoradiography. Black arrow = GST-C2 pro; white arrow = GST.
(D) Glutathione sepharose-bound GST-C2 pro V106A/H108A or V18A/L20A was prephosphorylated with [γ-32P]ATP and incubated in extract ± G6P. Samples were resolved by SDS-PAGE and detected by autoradiography. n = 3
(E) Flag-tagged full-length C2 WT or V106A/H108A mRNA was incubated in translationally-competent extract + 100 μM VDVAD-CHO. Translated flag-tagged C2 was retrieved with anti-flag agarose and samples were analyzed by immunoblotting with anti-flag.
(F) Percent survival of Xenopus oocytes microinjected with β-globin, full-length C2 WT, or V106A/H108A mRNA. Equivalent levels of C2 mRNA expression were determined as described in (E). n = 3
(G) Glutathione sepharose-bound GST-C2 pro was incubated in extract ± G6P and samples were retrieved over time and immunoblotted for PP1 and GST. n = 3
(H) Glutathione sepharose-bound GST-C2 pro was prephosphorylated with [γ-32P]ATP and incubated in buffer with His-PP1 pre-incubated in extract ± G6P or excess His-14-3-3ζ (27 μM). Samples were analyzed by SDS-PAGE and autoradiography. Black arrow = GST-C2 pro; white arrow = His-PP1. n = 4