GTP binding to the P-loop attenuates functional activity of DAPk. (A) Representative fluorescent images of HEK293T cells co-transfected with GFP and either DAPk WT, ΔP-loop or empty vector (pcDNA3). Insets show enlargements of boxed regions. (B) Quantitation of the extent of membrane blebbing and cell rounding seen in A. Each of the three DNA plasmids was transfected independently into three plates. A total of 300 cells were counted separately in each plate. Data represent mean±s.d. calculated from triplicates of 300 scored cells, P-values are calculated from Student's t-test. (C) HEK293T cells were co-transfected with GFP and either DAPk WT, ΔP-loop or luciferase (Luc), as a control, and the extent of cell detachment 24 h post-transfection, as a percentage of luciferase-transfected cells, was quantified. Data represent mean±s.d. (n=3), P-values are calculated from Student's t-test. Western blots in B and C show expression of DAPk constructs. All experiments were repeated at least three times with similar results. DAPk, death-associated protein kinase; GFP, green fluorescent protein; WT, wild type.