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

Figure 1.

Figure 1.

BCR-ABL1 induces phosphorylation of BTK in pre–B lymphoblastic leukemia cells. Protein expression and tyrosine phosphorylation of the pre–B cell receptor downstream signaling molecules SYK, SLP65, and BTK was studied by Western blot in three BCR-ABL1 + pre–B lymphoblastic leukemia cell lines (VII, VIII, and IX in Table I) in the presence or absence of STI571. The BTKpY223 antibody detected full-length BTK (77 kD) as well as two additional tyrosine phosphorylated proteins of 65-kD and 52-kD size (A–C). To confirm that constitutive phosphorylation of 77-kD full-length BTK is specific for BCR-ABL1 kinase activity, BTKY223-phosphorylation was studied in four BCR-ABL1–negative B cell precursor cell lines (B, left panel; see XI, XII, XIII, and XV in Table I). Dependence of BTK on BCR-ABL1 kinase activity also was confirmed in four primary cases of BCR-ABL1 + pre–B lymphoblastic leukemia, from which matched sample pairs before and during therapy with STI571 were available (B, right panel; cases I to IV in Table I). EIF-4E was used as a loading control. To determine whether Y223 phosphorylation of BTK is mediated by transphosphorylation through BCR-ABL1 or autophosphorylation, BCR-ABL1 + leukemia cells (IX; Table I) were treated for 24 h with the BCR-ABL1 kinase inhibitor, STI571, or the BTK inhibitor, LFM-A13 (C).