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Proline-rich region of CBL-Y371H mutant essential for enhanced signaling downstream of SCF-induced <t>c-KIT</t> activation. TF-1 cell lines expressing the indicated CBL mutant constructs were GM-CSF-deprived and then either left unstimulated (time 0) or stimulated with SCF (100 ng/ml) for the indicated time points (minutes). A–C, cell lysates were subjected to Western blotting for the indicated proteins: p-ERK, total ERK, and p-AKT (A) and total c-KIT and <t>p-c-KIT</t> (B). Shown is a representative experiment of two independent repeats with similar results. C, <t>phospho-c-KIT</t> signals in Western blottings were quantified using ImageJ analysis of scanned images, and the signals were first normalized to HSC-70 and then expressed as phospho-c-KIT signals relative to those in vector-transfected cells at 5 min, which were assigned a value of 1. Shown are pooled data of three independent repeats (mean ± S.E.). Red asterisks (*) indicate significance between CBL-Y371H and CBL-Y371-ΔPRR, and black asterisks (*) show significance between CBL-Y371H and vector cells (D). The indicated live TF-1 cell lines were analyzed by FACS for surface levels of c-KIT at various points after SCF stimulation. Mean fluorescence intensity relative to unstimulated controls is plotted. Shown are pooled data of three independent repeats. *, p < 0.05. E, THP-1 and GDM-1 cell lines were serum-deprived for 24 h and then left unstimulated or stimulated with SCF (100 ng/ml) for the indicated time points (minutes). Cell lysates were Western-blotted for phospho-c-KIT, total c-KIT, phospho-CBL, total CBL, and total CBL-B with HSC-70 used as a loading control. Shown is a representative of two independent repeats with similar results.
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Proline-rich region of CBL-Y371H mutant essential for enhanced signaling downstream of SCF-induced c-KIT activation. TF-1 cell lines expressing the indicated CBL mutant constructs were GM-CSF-deprived and then either left unstimulated (time 0) or stimulated with SCF (100 ng/ml) for the indicated time points (minutes). A–C, cell lysates were subjected to Western blotting for the indicated proteins: p-ERK, total ERK, and p-AKT (A) and total c-KIT and p-c-KIT (B). Shown is a representative experiment of two independent repeats with similar results. C, phospho-c-KIT signals in Western blottings were quantified using ImageJ analysis of scanned images, and the signals were first normalized to HSC-70 and then expressed as phospho-c-KIT signals relative to those in vector-transfected cells at 5 min, which were assigned a value of 1. Shown are pooled data of three independent repeats (mean ± S.E.). Red asterisks (*) indicate significance between CBL-Y371H and CBL-Y371-ΔPRR, and black asterisks (*) show significance between CBL-Y371H and vector cells (D). The indicated live TF-1 cell lines were analyzed by FACS for surface levels of c-KIT at various points after SCF stimulation. Mean fluorescence intensity relative to unstimulated controls is plotted. Shown are pooled data of three independent repeats. *, p < 0.05. E, THP-1 and GDM-1 cell lines were serum-deprived for 24 h and then left unstimulated or stimulated with SCF (100 ng/ml) for the indicated time points (minutes). Cell lysates were Western-blotted for phospho-c-KIT, total c-KIT, phospho-CBL, total CBL, and total CBL-B with HSC-70 used as a loading control. Shown is a representative of two independent repeats with similar results.

Journal: The Journal of Biological Chemistry

Article Title: Structural Determinants of the Gain-of-Function Phenotype of Human Leukemia-associated Mutant CBL Oncogene

doi: 10.1074/jbc.M116.772723

Figure Lengend Snippet: Proline-rich region of CBL-Y371H mutant essential for enhanced signaling downstream of SCF-induced c-KIT activation. TF-1 cell lines expressing the indicated CBL mutant constructs were GM-CSF-deprived and then either left unstimulated (time 0) or stimulated with SCF (100 ng/ml) for the indicated time points (minutes). A–C, cell lysates were subjected to Western blotting for the indicated proteins: p-ERK, total ERK, and p-AKT (A) and total c-KIT and p-c-KIT (B). Shown is a representative experiment of two independent repeats with similar results. C, phospho-c-KIT signals in Western blottings were quantified using ImageJ analysis of scanned images, and the signals were first normalized to HSC-70 and then expressed as phospho-c-KIT signals relative to those in vector-transfected cells at 5 min, which were assigned a value of 1. Shown are pooled data of three independent repeats (mean ± S.E.). Red asterisks (*) indicate significance between CBL-Y371H and CBL-Y371-ΔPRR, and black asterisks (*) show significance between CBL-Y371H and vector cells (D). The indicated live TF-1 cell lines were analyzed by FACS for surface levels of c-KIT at various points after SCF stimulation. Mean fluorescence intensity relative to unstimulated controls is plotted. Shown are pooled data of three independent repeats. *, p < 0.05. E, THP-1 and GDM-1 cell lines were serum-deprived for 24 h and then left unstimulated or stimulated with SCF (100 ng/ml) for the indicated time points (minutes). Cell lysates were Western-blotted for phospho-c-KIT, total c-KIT, phospho-CBL, total CBL, and total CBL-B with HSC-70 used as a loading control. Shown is a representative of two independent repeats with similar results.

Article Snippet: Anti-phospho-ERK1/2 (pERK1/2), anti-ERK1/2 (ERK1/2), anti-phospho-AKT (pAKT), anti-AKT (tAKT), anti-phospho-c-KIT (p-c-KIT), anti-c-KIT anti-phospho-CBL (Tyr-700), and anti-phospho-CBL (Tyr-774) were obtained from Cell Signaling Technologies.

Techniques: Mutagenesis, Activation Assay, Expressing, Construct, Western Blot, Plasmid Preparation, Transfection, Fluorescence

Proline-rich region of CBL-Y371H is required for hyper-activation of basal and SCF-induced signaling. A, TF-1 cell lines expressing indicated CBL mutant constructs were GM-CSF deprived and either left unstimulated or stimulated with SCF for the indicated time points. Anti-HA immunoprecipitations were carried out followed by anti-Tyr(P), anti-CBL-Tyr(P)-700, or anti-CBL-Tyr(P)-774 immunoblotting to visualize the phosphorylation of ectopic overall and site-specific phosphorylation of CBL proteins. Anti-HA blotting shows comparable loading. Shown is a representative experiment of two independent repeats with similar results. B, GM-CSF-deprived TF-1 cells expressing CBL-Y371H were stimulated with SCF for 15 min, and 5 mg of cell lysate protein aliquots used for pulldown with 50 μg of GST (lane 1, control), GST-CBL-N (lane 2), or GST-CBL-C (lane 3) fusion proteins non-covalently bound to glutathione-Sepharose beads. After washing, the bound proteins were visualized anti-Tyr(P) (4G10) immunoblotting, and the membrane was re-probed with an anti-phospho-c-KIT antibody. The whole cell lysate (25 μg) was concurrently resolved (lane 4). The molecular mass markers (in kilodaltons) are indicated on left. C, antibody array analysis of phosphoproteins in whole cell lysates. TF-1 cells expressing empty vector, CBL-Y371H, or CBL-Y371H-ΔPRR were GM-CSF-deprived and stimulated with SCF for 15 min. Cell lysates were collected in the antibody array lysis buffer supplied with the kit and incubated with antibody array filters and developed using enhanced chemiluminescence, followed by densitometry analysis of phosphoprotein signals. Signals were expressed relative to those in unstimulated vector control TF-1 cell lysates. D, antibody array analysis of preferential binding of cellular phosphoproteins with PRR-containing GST-CBL-C versus GST-CBL-N (TKB domain). TF-1 cells expressing CBL-Y371H were GM-CSF-deprived and stimulated with SCF for 15 min. 5-mg aliquots of cell lysate protein were incubated with 50 μg of glutathione-Sepharose-bound GST-CBL-N or GST-CBL-C fusion proteins. Bound proteins were eluted in 1% SDS-containing lysis buffer by a 5-min incubation at 95 °C, cooled to room temperature, diluted 10-fold in antibody array buffer, and analyzed for phosphoprotein signals using antibody arrays as in C. Data are presented as a ratio of GST-CBL-C pulldown signals over GST-CBL-N pulldown signals for each phosphoprotein. Boxes indicate preferential binding to GSCT-CBL-C or GST-CBL-N.

Journal: The Journal of Biological Chemistry

Article Title: Structural Determinants of the Gain-of-Function Phenotype of Human Leukemia-associated Mutant CBL Oncogene

doi: 10.1074/jbc.M116.772723

Figure Lengend Snippet: Proline-rich region of CBL-Y371H is required for hyper-activation of basal and SCF-induced signaling. A, TF-1 cell lines expressing indicated CBL mutant constructs were GM-CSF deprived and either left unstimulated or stimulated with SCF for the indicated time points. Anti-HA immunoprecipitations were carried out followed by anti-Tyr(P), anti-CBL-Tyr(P)-700, or anti-CBL-Tyr(P)-774 immunoblotting to visualize the phosphorylation of ectopic overall and site-specific phosphorylation of CBL proteins. Anti-HA blotting shows comparable loading. Shown is a representative experiment of two independent repeats with similar results. B, GM-CSF-deprived TF-1 cells expressing CBL-Y371H were stimulated with SCF for 15 min, and 5 mg of cell lysate protein aliquots used for pulldown with 50 μg of GST (lane 1, control), GST-CBL-N (lane 2), or GST-CBL-C (lane 3) fusion proteins non-covalently bound to glutathione-Sepharose beads. After washing, the bound proteins were visualized anti-Tyr(P) (4G10) immunoblotting, and the membrane was re-probed with an anti-phospho-c-KIT antibody. The whole cell lysate (25 μg) was concurrently resolved (lane 4). The molecular mass markers (in kilodaltons) are indicated on left. C, antibody array analysis of phosphoproteins in whole cell lysates. TF-1 cells expressing empty vector, CBL-Y371H, or CBL-Y371H-ΔPRR were GM-CSF-deprived and stimulated with SCF for 15 min. Cell lysates were collected in the antibody array lysis buffer supplied with the kit and incubated with antibody array filters and developed using enhanced chemiluminescence, followed by densitometry analysis of phosphoprotein signals. Signals were expressed relative to those in unstimulated vector control TF-1 cell lysates. D, antibody array analysis of preferential binding of cellular phosphoproteins with PRR-containing GST-CBL-C versus GST-CBL-N (TKB domain). TF-1 cells expressing CBL-Y371H were GM-CSF-deprived and stimulated with SCF for 15 min. 5-mg aliquots of cell lysate protein were incubated with 50 μg of glutathione-Sepharose-bound GST-CBL-N or GST-CBL-C fusion proteins. Bound proteins were eluted in 1% SDS-containing lysis buffer by a 5-min incubation at 95 °C, cooled to room temperature, diluted 10-fold in antibody array buffer, and analyzed for phosphoprotein signals using antibody arrays as in C. Data are presented as a ratio of GST-CBL-C pulldown signals over GST-CBL-N pulldown signals for each phosphoprotein. Boxes indicate preferential binding to GSCT-CBL-C or GST-CBL-N.

Article Snippet: Anti-phospho-ERK1/2 (pERK1/2), anti-ERK1/2 (ERK1/2), anti-phospho-AKT (pAKT), anti-AKT (tAKT), anti-phospho-c-KIT (p-c-KIT), anti-c-KIT anti-phospho-CBL (Tyr-700), and anti-phospho-CBL (Tyr-774) were obtained from Cell Signaling Technologies.

Techniques: Activation Assay, Expressing, Mutagenesis, Construct, Western Blot, Ab Array, Plasmid Preparation, Lysis, Incubation, Binding Assay