ARG tyrosine kinase activity is inhibited by STI571 by Keiko Okuda, Ellen Weisberg, D. Gary Gilliland, and James D. Griffin Blood Volume 97(8):2440-2448 April 15, 2001 ©2001 by American Society of Hematology

Comparison of fusion genes Comparison of fusion genes.(A) Schematic representation of fusion genes. Comparison of fusion genes.(A) Schematic representation of fusion genes. PNT indicates pointed domain; KD, tyrosine kinase domain; DNA, DNA-binding domain; ABD, actin-binding domain; tm, transmembrane domain; SH3: SRC homology 3; SH2, SRC homology 2. (B) Comparison of kinase domains of ARG, ABL, PDGFR, KIT, and JAK2. Keiko Okuda et al. Blood 2001;97:2440-2448 ©2001 by American Society of Hematology

Comparison of cellular tyrosine phosphorylation patterns in fusion protein-transfected Ba/F3 cells and untransfected Ba/F3 cells.(A) Comparison of cellular tyrosine phosphorylation patterns in Ba/F3.p210 cells, TEL/ARG Ba/F3 cells, and TEL/ABL Ba/F3 cells. Comparison of cellular tyrosine phosphorylation patterns in fusion protein-transfected Ba/F3 cells and untransfected Ba/F3 cells.(A) Comparison of cellular tyrosine phosphorylation patterns in Ba/F3.p210 cells, TEL/ARG Ba/F3 cells, and TEL/ABL Ba/F3 cells. Anti–β-actin was used as a loading control. The first and second lanes shown (Ba/F3.p210 and TEL/ARG Ba/F3, respectively) are derived from the same Western blot; the third lane shown (TEL/ABL Ba/F3) is derived from an independent Western blot performed in parallel with the first blot. (B) Comparison of cellular tyrosine phosphorylation patterns in Ba/F3.p210 cells and Ba/F3 cells. An anti-ABL antibody was used to measure levels of ABL in both cell types and to show the expression of BCR/ABL in Ba/F3.p210 cells. WB indicates Western blot. Keiko Okuda et al. Blood 2001;97:2440-2448 ©2001 by American Society of Hematology

Inhibition of TEL/ARG autophosphorylation by STI571 Inhibition of TEL/ARG autophosphorylation by STI571.TEL/ARG was immunoprecipitated from TEL/ARG-transfected Ba/F3 cells cultured in the absence or presence of STI571 for 24 hours (lanes 1 and 2, respectively). Inhibition of TEL/ARG autophosphorylation by STI571.TEL/ARG was immunoprecipitated from TEL/ARG-transfected Ba/F3 cells cultured in the absence or presence of STI571 for 24 hours (lanes 1 and 2, respectively). An anti-PAC1 polyclonal goat antibody was used to immunoprecipitate the phosphatase PAC1 as a nonspecific control (lane 3). Protein lysate from TEL/ARG-Ba/F3 cells was also incubated in the presence of protein G beads alone, as a nonspecific control (lane 4). Immunoblotting was performed on all samples with anti-pTYR (upper panel) and anti-ARG (lower panel). Whole cell lysates from TEL/ARG-Ba/F3 cells cultured in the absence and presence of STI571 (lanes 6 and 7, respectively) were run in parallel with the immunocomplexes, and immunoblotting was similarly performed on these samples with anti-pTyr (upper panel) and anti-ARG (lower panel). Whole cell lysate from Ba/F3.p210 cells was run alongside whole cell lysates from TEL/ARG-Ba/F3 cells for comparison (lane 5). IP indicates immunoprecipitation. Keiko Okuda et al. Blood 2001;97:2440-2448 ©2001 by American Society of Hematology

Comparison of the effects of STI571 on cellular tyrosine phosphorylation in Ba/F3 cells expressing BCR/ABL, TEL/ABL, TEL/ARG, TEL/PDGFR, or TEL/JAK2.(A) An anti-pTYR immunoblot showing cellular tyrosine phosphorylation in untransfected Ba/F3 cells and TEL/A... Comparison of the effects of STI571 on cellular tyrosine phosphorylation in Ba/F3 cells expressing BCR/ABL, TEL/ABL, TEL/ARG, TEL/PDGFR, or TEL/JAK2.(A) An anti-pTYR immunoblot showing cellular tyrosine phosphorylation in untransfected Ba/F3 cells and TEL/ARG-Ba/F3 cells cultured in the presence and absence of STI571 (left panel). An anti-pTYR immunoblot showing cellular tyrosine phosphorylation in untransfected Ba/F3 cells, TEL/ABL-Ba/F3, and Ba/F3.p210 cells cultured in the presence and absence of STI571 (right panel). Immunoblots were stripped and incubated with anti–β-actin as a loading control. (B) Effect of 24-hour STI571 treatment on viability of cells analyzed in part A. (C) Anti-pTYR immunoblots showing cellular tyrosine phosphorylation in TEL/ABL-Ba/F3, TEL/ARG-Ba/F3, Ba/F3.p210, TEL/PDGFR-Ba/F3, TEL/JAK2-Ba/F3, and untransfected Ba/F3 cells treated with increasing concentrations of STI571. Immunoblots were stripped and incubated with anti–β-actin as a loading control. Keiko Okuda et al. Blood 2001;97:2440-2448 ©2001 by American Society of Hematology

Comparison of the effects of STI571 on cellular proliferation and viability in Ba/F3 cells expressing BCR/ABL, TEL/ARG, TEL/ABL, TEL/PDGFR, or TEL/JAK2.(A) Growth of cells cultured in the presence of increasing concentrations of STI571 (0.01-5 μM) for 24 ho... Comparison of the effects of STI571 on cellular proliferation and viability in Ba/F3 cells expressing BCR/ABL, TEL/ARG, TEL/ABL, TEL/PDGFR, or TEL/JAK2.(A) Growth of cells cultured in the presence of increasing concentrations of STI571 (0.01-5 μM) for 24 hours. (B) Growth of cells cultured in the presence of increasing concentrations of STI571 (0.01-5 μM) for 48 hours. (C) Growth of cells cultured in the presence of increasing concentrations of STI571 (0.01-5 μM) for 72 hours. For all cell lines, values obtained for the 24-hour time points were calculated as the average of 3 independent experiments, shown as percentage untreated (control) cells. Values obtained for the 48- and 72-hour time points, respectively, were calculated as the average of 2 independent experiments, shown as percentage untreated (control) cells. Each cell line is represented by a different symbol, as shown in the figure legends. Keiko Okuda et al. Blood 2001;97:2440-2448 ©2001 by American Society of Hematology

Effects of STI571 on cell viability and apoptosis in Ba/F3 cells expressing TEL/ARG and untransfected Ba/F3 cells, as determined by annexin-PI staining.(A) Eighteen-hour treatment of TEL/ARG-Ba/F3 cells with vehicle (left panel) or 1 μM STI571 (right panel). Effects of STI571 on cell viability and apoptosis in Ba/F3 cells expressing TEL/ARG and untransfected Ba/F3 cells, as determined by annexin-PI staining.(A) Eighteen-hour treatment of TEL/ARG-Ba/F3 cells with vehicle (left panel) or 1 μM STI571 (right panel). (B) Twenty-eight–hour treatment of TEL/ARG-Ba/F3 cells with vehicle (left panel) or 1 μM STI571 (right panel). (C) Twenty-eight–hour treatment of untransfected Ba/F3 cells with vehicle (left panel) or 1 μM STI571 (right panel). FITC indicates fluorescein isothiocyanate. Keiko Okuda et al. Blood 2001;97:2440-2448 ©2001 by American Society of Hematology

IL-3 rescue of TEL/ARG-Ba/F3 cells and TEL/ABL-Ba/F3 cells IL-3 rescue of TEL/ARG-Ba/F3 cells and TEL/ABL-Ba/F3 cells.(A) Growth curves for TEL/ARG-Ba/F3 cells treated with STI571 for 24 (░) and 72 hours (▪), respectively, and cultured in the absence or presence of 15% WEHI-conditioned medium, used as a source of I... IL-3 rescue of TEL/ARG-Ba/F3 cells and TEL/ABL-Ba/F3 cells.(A) Growth curves for TEL/ARG-Ba/F3 cells treated with STI571 for 24 (░) and 72 hours (▪), respectively, and cultured in the absence or presence of 15% WEHI-conditioned medium, used as a source of IL-3 (left panel). Growth curves for TEL/ABL-Ba/F3 cells treated as described for TEL/ARG-Ba/F3 cells (right panel). Experiments were performed in duplicate for each cell line, and viability counts were determined by trypan blue exclusion and shown as percentage control. (B) Annexin-PI analysis of TEL/ARG-Ba/F3 cells (left panel) and TEL/ABL-Ba/F3 cells (right panel), treated as described in panel A. Keiko Okuda et al. Blood 2001;97:2440-2448 ©2001 by American Society of Hematology