Signaling responses induced by anti-PtC and anti-Sm BCRs on stimulation with external antigen. (A) Flow cytometry analysis of Ca²⁺ flux after stimulation of TKO cells expressing a leukemia-derived anti-PtC or a leukemia-derived anti-Sm BCR. Stimulations were done with the corresponding cognate antigen or with an anti-LC antibody. (B) Flow cytometry analysis of Ca²⁺ flux in primary leukemic cells expressing a transgenic anti-PtC or a transgenic anti-Sm BCR. The cells were stimulated with antigen, anti-IgM, or Ionomycin (Ion.) as indicated in the figure. Ionomycin was used as a positive control. (C) Phospho-flow analysis of Eμ-TCL1-derived leukemias expressing an anti-PtC or an anti-Sm BCR. Cells were stimulated for 10 minutes at 37°C with their cognate antigen or with anti-IgM. Blue lines indicate the phospho-SYK (pSYK) and phospho-BLNK (pBLNK) signal in cells stimulated with cognate antigen, red lines in cells stimulated with anti-IgM, and filled histograms in unstimulated cells. These results are representative of 7 experiments performed with leukemias expressing an anti-PtC and 5 experiments performed with leukemias expressing an anti-Sm BCR. The remaining experiments are shown in supplemental Figure 4. (D) Immunoblotting analysis of cellular extracts from primary leukemic cells expressing an anti-PtC or an anti-Sm BCR. Cells were stimulated for 10 minutes with cognate antigen or were incubated for 2 hours with 1 μM R406 prior to lysis. The levels of pSYK are not reduced by R406 treatment because the investigated Y352 residue is phosphorylated by SRC-family kinases.