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. 2005 Jun 6;201(11):1837–1852. doi: 10.1084/jem.20042101

Figure 7.

Figure 7.

BTKp52 facilitates BTK- and BCR-ABL1–dependent activation of PLCγ1 and STAT5 through its SH3 domain. BCR-ABL1, full-length BTK, BTKp52, and the SH3-domain of BTK were expressed in 293T embryonic kidney cells alone or in various combinations in the presence or absence of STI571 or LFM-A13. As a readout, cells were harvested, subjected to intracellular staining for tyrosine-phosphorylated PLCγ1 or STAT5, and analyzed by flow cytometry (A). To visualize cytoplasmic localization of tyrosine-phosphorylated PLCγ1 and nuclear localization of activated STAT5, the stained cells also were subjected to analysis by confocal laser microscopy (B). 106 293T cells were transfected transiently with full-length BTK for 24 h and subjected to immunoprecipitation of full-length BTK. Immunoprecipitation (IP) was controlled by a BTK-specific Western blot (C). Kinase activity of immunoprecipitated BTK was analyzed in an in vitro kinase assay using 150 ng of a PLCγ1 fragment (amino acids 530 to 850) as substrate. In parallel, 25 ng of recombinant active BTK and 100 ng of kinase-deficient SH3-domain of BTK were used in kinase assays as positive and negative controls, respectively.