Figure 2

ARTC2.1 is upregulated on microglia upon stimulation with LPS and U0126. (a) Mixed glial cells were stimulated with or without LPS (0.1 µg/ml), U0126 (10 µM) or both for 24 h, followed by incubation with eNAD⁺ (50 µM) and DTT (2.5 mM) for 15 min. Etheno-ADP-ribosylated cell surface proteins were detected with mAb 1G4 (displayed as mean fluorescence intensity, MFI). (b) Mixed glial cell cultures were stimulated with LPS (0.1 µg/ml) and U0126 (10 µM) for 24 h. Total cells (left) or FACS sorted astrocytes and microglia (right) were then incubated for 15 min with radioactive ³²P-NAD⁺ (1 µM) in the presence or absence of DTT (2.5 mM). Cells were lysed, proteins were size fractioned by SDS PAGE and subjected to autoradiography. (c) Mixed glial cells from BALB/c WT and ARTC2.1^−/− mice were stimulated with LPS (0.1 µg/ml) and U0126 (10 µM) for 24 h, and then incubated with eNAD⁺ (50 µM) and DTT (2.5 mM) for 15 min. Etheno-ADP-ribosylated surface proteins were detected with mAb 1G4. (d) mRNA levels of Art2a from FACS sorted microglia (n = 5 individual experiments) of unstimulated or LPS/U0126 stimulated mixed glial cell cultures were determined by quantitative real-time PCR. (e) Surface expression of ARTC2.1 by microglia of LPS/U0126 stimulated or control mixed glial cell cultures of BALB/c WT or ARTC2^−/− mice was analyzed by flow cytometry as in Fig. 1c. Data are representative of 2–3 independent experiments.