Membrane compartmentalization restricts the specificity of target protein (etheno)–ADP-ribosylation by ART2.2. (A-B) Untransfected (Untr.), ART2.2-GPI–transfected (GPI), and ART2.2-Tm–transfected (Tm) DC27.10 cells were incubated for 10 minutes in the presence of 1 μM (A) or 50 μM etheno-NAD (B). Cells were washed and then lysed in 2 steps in 1% TX-100 for 10 minutes at 4°C and then for 20 minutes at 37°C, as in Figure 3. TX-100–resistant pellets (P) were solubilized by sonication in 2% SDS. Proteins in cell lysates were acetone precipitated and subjected to size fractionation by SDS-PAGE, followed by Western blotting onto PVDF membranes. Immunodetection was performed as in Figure 6. (C) Cells were lysed by TX-100 and SDS, as described in panel B. Then 1 μM etheno-NAD was added to each lysate, and lysates were incubated for 10 minutes at 37°C. Reactions were stopped by the addition of acetone, and proteins were analyzed by immunoblotting as in panel A. (D) ART2.2-GPI–transfected DC27.10 cells were incubated for 10 minutes in the presence of 1 μM ³²P-NAD. Cells were washed and lysed in 2 steps in 0.05% TX-100 (lanes 1, 2) or in 1% TX-100 (lanes 3, 4) for 10 minutes at 4°C and then in 1% TX-100 for 20 minutes at 37°C. Lysates were subjected to immunoprecipitation with immobilized anti–LFA-1 or anti-P2X7. Total protein in cell lysates was visualized by Coomassie brilliant blue staining, and ART2.2 was visualized by immunoblotting with PO-conjugated anti-FLAG mAb M2 (left panels). Radioactivity incorporated into proteins was determined by autoradiography; exposure times were 6 hours for LFA-1, 6 hours for total proteins, and 36 hours for P2X7 (right panels).