Figure 2
From: A pathway sensor for genome-wide screens of intracellular proteolytic cleavage
Design of a GLUC-based caspase sensor. (a) Schematic representation of Actin-dN, DEVDG2F, FDEVDG2, DEVDG3 and DEVAG2F. Actin, grey box; dNGLUC, shaded box. (b) Activation of FDEVDG2 by caspase 8 and 9. 293ET cells were co-transfected with 500 ng of Actin-dN or FDEVDG2 and the indicated amounts of caspase 8 (left panel) or caspase 9 (right panel) in a 12-well plate. SN was tested for GLUC activity after 30 h. Error bars were calculated from three independent transfections. RLU, relative light units. (c) Immune-blotting of cleaved FDEVDG2. Transiently transfected 293ET cells expressing FDEVDG2 together with GFP or caspase 9 were grown for 30 h prior to cell lysis. Lysates were resolved by 10% PAGE and immune-blots were analyzed with anti-Flag M2 (upper panel) or anti-GLUC (lower panel) antibody. Full-length FDEVDG2 migrates at 62 kDa (marked by an asterisk) and caspase 9-cleaved Actin-FDEVDG2 migrates around 46 kDa (marked by an arrow).