Figure 5. Association of G-actin with PPP1R15 regulates eIF2α phosphatase activity.
(A) Immunoblot for phosphorylated eIF2α (P-eIF2α), total eIF2α, and actin. Wild-type (WT) mouse embryonic fibroblasts (MEF) were treated with jasplakinolide 1 µM for the indicated times. Lysates were subjected to sedimentation assay and immunoblot for G-actin in the supernatant (G) or F-actin in the pellet (F). (B) Immunoblot for phosphorylated eIF2α (P-eIF2α), total eIF2α, and actin. WT MEFs were treated with the indicated concentrations of jasplakinolide for 1 hr. Lysates were analysed as in ‘A’. (C) Immunoblot for phosphorylated eIF2α (P-eIF2α), total eIF2α, and PPP1R15A. WT MEFs were treated with thapsigargin 400 nM for the indicated times, without or with jasplakinolide 1 µM. (D) Immunoblot for P-eIF2α and PPP1R15A (hR15A). GFP-hPPP1R15A Tet-On HeLa cells were treated with doxycycline to induce transgene expression and then with thapsigargin 400 nM for the indicated times. Cells were co-treated with jasplakinolide or latrunculin B 1 µM or vehicle as indicated. Accompanying graphs show means ±SEM of n = 3 independent repeats.
Figure 5—figure supplement 1. Immunoblot for phosphorylated eIF2α (P-eIF2α), total eIF2α and PPP1R15A.
