FIGURE 2.

GDP-ribosyl cyclase activity and CD38 phosphorylation in ABA-stimulated N9 cells. A, after addition of the indicated concentrations of ABA to N9 cells (3 × 10⁷), the GDP-ribosyl cyclase activity was determined on intact cells (black bars), as described under “Experimental Procedures.” The cells were also preincubated with I-PKA (1 μm for 90 min) before the addition of 20 μm ABA (white bar). The results are expressed as cGDPR production relative to untreated controls (2.5 ± 0.7 pmol cGDPR/min/mg protein) and are the means ± S.D. of at least five experiments. B, CD38 was immunopurified (see “Experimental Procedures”) from cells preincubated for 10 min without (lanes a and c) or with 20 μm ABA (lanes b and d). Western blots were stained with an anti-phosphoserine mAb (lanes a and b) or with an anti-CD38 mAb (lanes c and d). The graph displayed below the Western blots shows the bioluminescence intensity of the anti-phosphoserine mAb-stained bands, as quantified using the Chemidoc System; the values are expressed as optical density/mm² and are the means ± S.D. of three experiments.