Disruption of lipid rafts by β-MCD inhibits the activity of ART2.2-GPI. (A) DC27.10 cells stably transfected with ART2.GPI (GPI; filled symbols) or ART2.2-Tm (Tm; open symbols) were incubated for 60 minutes in the absence (diamonds) or presence (squares) of 10 mM MCD. Cells were washed and incubated further in the presence of 1 μM etheno-NAD for the indicated times. Cell-surface protein ADP-ribosylation was detected by FACS analysis with Alexa488-1G4, as in Figure 5. (B) Equal aliquots of ART2.2-GPI and ART2.2-TM cells were mixed and incubated for 60 minutes in the absence or presence of 5 to 10 mM MCD. Cells were then lysed in 2 steps in 1% TX-100, and proteins in cleared lysates were analyzed by immunoblot analysis, as in Figure 3. (C) Cells were incubated for 60 minutes in the absence or presence of 10 mM MCD. Cells were then washed and incubated further for 10 minutes in the presence of 1 μM etheno-NAD. Immunodetection was performed by incubation of blots with mouse anti–etheno-adenosine mAb 1G4 followed by PO-conjugated anti–mouse IgG 1G4. Bound antibody was detected using ECL.