Characterization of GFP-tagged PARP1 expression in PARP1-deficient cells. a, schematic diagram of GFP fusion constructs. The human PARP1 was fused at either its N terminus or C terminus to GFP. b, constructions allowing expression of PARP1 fused to GFP were transiently expressed in PARP1-deficient (A1) cells. Total extracts from wild-type (C3H10T½), PARP1-deficient (A1), and GFP-tagged PARP1-expressing cells were prepared and immunoblotted with anti-PARP1 antibody. Actin blots are shown as a loading control. c, GFP image obtained at the onset of the time lapse experiment. Bar, 10 μm. d, cellular localization was also analyzed by indirect immunofluorescence microscopy using anti-PARP1 monoclonal antibody in order to detect both PARP1-GFP in knock-out cells (A1) (left) and endogenous PARP1 in normal cells (C3H10T½) (right). DNA was stained with Hoechst 33342. e, for the determination of PARP1 expression in mouse (C3H10T½), human (SK-N-SH), and PARP1-deficient cells expressing a functional PARP1-GFP construct, multiple fields were examined to count at least 200 cells per cell line. The graph presents the integrated fluorescence intensity of nucleus stained with monoclonal antibody (clone C2-10) that recognizes endogenous PARP1. eGFP, enhanced GFP.
