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. 2011 Jul 8;12(9):917–923. doi: 10.1038/embor.2011.126

Figure 3.

Figure 3

GTP binding to the P-loop promotes in cis Ser 308 negative autophosphorylation. (A) HEK293T cells transfected with Flag–DAPk WT, ΔP-loop or T701N mutants were analysed by western blot with phosphoSer 308(pSer 308)-specific and Flag antibodies. Actin was used as a loading control. (B) HEK293T cells were co-transfected with catalytically inactive DAPk (HA–K42A) and increasing amounts of GFP–DAPk, and analysed by western blot with antibodies against total DAPk and pSer 308. Actin was used as a loading control. (C) Flag–DAPk WT or the indicated mutants were immunoprecipitated from HEK293T cells with Flag antibodies and blotted for Flag and co-precipitating endogenous PP2A B’ subunit. (D) HEK293T cells were co-transfected with Flag–DAPk WT or ΔP-loop mutant, 24 h post-transfection cells were treated with 100% ethanol or 100 nM okadaic acid for 50 min. Where indicated, 10 min after okadaic-acid addition, cells were treated with 10 μM ionomycin for the remaining time. DAPk proteins were analysed by western blot with Flag and pSer 308 antibodies. Actin was used as a loading control. DAPk, death-associated protein kinase; GFP, green fluorescent protein; HA, haemagglutinin; IP, immunoprecipitation; ROC, Ras of complex proteins; WT, wild type.