CD38 signaling is dependent on the presence of ZAP-70. (A) CD38 signaling benefits from the presence of ZAP-70. BJAB–ZAP-70⁺ and mock-transfected cells were treated with the indicated antibodies, and the lysates were immunoprecipitated and blotted using antiphosphotyrosine antibodies. H and L chain refer to the heavy and light chains of the precipitating Ab. (B) Total protein tyrosine phosphorylation profile of 3 patients with CLL upon CD38 or IgM ligation (5 minutes, 37°C). CLL no. 21 and no. 27 were CD38⁺/ZAP-70⁺, while no. 47 was CD38⁺/ZAP-70⁻. The arrow shows a band likely corresponding to ERK1/2. No increase in protein tyrosine phosphorylation was observed with CLL no. 47 cells. (C) Analysis of ERK1/2 phosphorylation in a subset of 12 patients with CLL upon CD38 and IgM ligation. CD38⁺/ZAP-70⁺ cells show a clear increase in phosphorylation compared with CD38⁺/ZAP-70⁻ cells. (D) ERK1/2 phosphorylation was studied in 26 patients with CLL; gels were scanned and the band intensities were measured by the ImageJ software and plotted by dividing the patients according to different molecular parameters. The only statistically significant difference is observed by dividing CD38⁺ patients (n = 17) on the basis of ZAP-70 expression (left panel). No difference is scored when using IgV mutations (right panel). Data are expressed as percentage of increase in ERK1/2 phosphorylation compared with background; the horizontal line represents the mean of all samples. Statistical difference was calculated according to the Student 2-sample, 2-tailed t test.