Effect of SPRY2 on BCR signaling. To study the role of SPRY2 in CLL and nB cells, we isolated CLL and nB cells from patients and healthy donors, respectively. (A) nB cells, primary human CLL cells, and Mec-1 CLL cells were stimulated by BCR crosslinking for 0, 6, 12, 18, 24, and 48 hours. Cells were washed and protein lysate was prepared. Protein level of SPRY2 was determined by western blotting. SPRY2 levels in nB cells (top), SPRY2 levels in primary CLL cells from patient (middle), and SPRY2 levels in Mec-1 CLL cells (bottom) are shown. (B) To test the efficacy of siRNAs against SPRY2, nB cells were transfected with siRNAa and siRNAb after 48 hours of transfection cells, and were washed and lysate was prepared. An equal amount of protein was loaded in each well, and scramble siRNA and β-actin was used as control. Displayed is scanned western blot showing decrease in SPRY2 levels after siRNA treatment. (C) nB cells were isolated from healthy donors and nucleofected with scramble, siRNA A, and siRNA B. After 48 hours, calcium influx assay was performed using Indo-1 dye dots representing 20-second time intervals. Displayed is mean graph of Indo-1 violet/Indo-1 blue ratio, nB-cell samples from different healthy donors (n = 5), and P < .0001.