CD38 increases polarized RasGRP2 localization. Cells were stained with anti-RasGRP2 antibody followed by Alexa Fluor 546–conjugated anti-rabbit antibody and imaged by confocal microscopy. F-actin and nuclei were stained with Alexa Fluor 488–conjugated phalloidin and DAPI, respectively. (A) Representative images of RasGRP2 localization in MEC1-GFP and MEC1-CD38H cells. Examples of cells with polarized RasGRP2 localization are indicated with an asterisk. Scale bar, 20 µm. (B) Fluorescence intensity profile of RasGRP2 obtained along the longitudinal axis of 2 representative MEC1-GFP and MEC1-CD38H cells. Scale bar, 5 µm. (C) The percentage of cells with polarized RasGRP2 localization was quantified by scoring n ≥ 100 cells in each of the 3 independent experiments. **P < .01, determined by 2-tailed Student t test. (D-F) RasGRP2 localization in CD38^high and CD38^low CLL patient samples. (D) Representative images of one CD38^high (CLL 2) and one CD38^low (CLL 5) CLL sample are shown. Scale bar, 10 μm. (E) The RasGRP2 polarization index, indicating distribution of the fluorescence intensity, was calculated using ImageJ Oval Profile plug-in (supplemental Data). Graph shows the mean values obtained for each CLL patient sample (n ≥ 30 cells analyzed per sample), horizontal bars indicate mean values of the RasGRP2 polarization index obtained for the 2 groups by grouping CD38^low (n = 7, green) and CD38^high (n = 7 red) samples. **P < .01, determined by 2-tailed Student t test.