Figure 1
Immunoprecipitation and mass spectrometry analyses identify CD25 as a major ADP-ribosylation target on YAC-1 cells.
(a) YAC-1 cells were stained with fluorochrome-conjugated mAbs specific for ARTC2.2 and CD25 and analyzed by flow cytometry. (b) YAC-1 cells were incubated with 32P-NAD+. Cell lysates were subjected to immunoprecipitation, proteins were size fractionated by SDS-PAGE and incorporated radioactivity was detected by autoradiography. Lanes 1, 2: cell lysates before and after precipitation of CD25; lane 3: control precipitation with protein G, lane 4: precipitation with anti-CD25 (PC61). (c–e) YAC-1 cells were incubated with etheno-NAD+. Etheno-ADP-ribosylated proteins were purified from the cell lysate using an affinity column with etheno-adenosine specific mAb 1G4. Bound proteins were eluted in four fractions with etheno-adenosine. Proteins in the cell lysate (lane 1), column flow through (lane 2), wash (lane 3) and eluates (lanes 4–7) were size fractionated by SDS-PAGE. Total protein was visualized by Coomassie staining (c), etheno-ADP-ribosylated proteins were visualized in a parallel immunoblot analysis with mAb 1G4 (d). The 50 kD band in lane 5 (panel c) was excised from the gel and subjected to trypsin digestion and nanospray mass spectrometry. MALDI-TOF spectrum of a fragmented tryptic peptide of 1892,9 kD (e). Boxed numbers indicate the masses of individual peptide peaks, numbers above brackets indicate the mass differences between adjacent peptide peaks. The amino acid sequence of the N-terminal peptide of CD25 is shown on top with numbers corresponding to the masses of the respective peptide fragments. Results are representative of three independent experiments.
