Figure 5
Detection of ADP-ribosylated proteins by flow cytometry. C-33A cells (1 × 107/ml) transiently transfected with a Flag-tagged chicken ART4 containing plasmid (A, B) or the empty plasmid (pSecTag B) (C, D) were incubated at 37°C in the presence or absence of 200 μM etheno-NAD+ for 30 min. After washing, cells were stained with an etheno-adenosine specific antibody (1G4) (A, C) or an anti-Flag antibody (M2) (B, D) and the respective isotype control. Numbers in the upper right quadrant indicate the percentage of fluorescence positive cells. Shown are typical scattergrams (fluorescence (FL1) vs. forward scatter (FSC)) of one representative experiment out of three. (E) Chicken erythrocytes (1% v/v), isolated by density gradient centrifugation, were treated as described above and stained with the 1G4 antibody. Shown is one representative experiment out of 8.