Figure 2
Figure 2. Functional effects of NOTCH2 and FLT3 splice variants. (A) This figure displays unsupervised clustering analyses results obtained from the TaqMan gene expression assays of Notch2 target genes HES1, DXT1 and HEY1 carried out in 49 AML patients expressing NOTCH2-FL, FLT3-FL, and their splice variants. Expression levels of NOTCH2-FL, FLT3-FL, and their splice variants were determined by RT-PCR and DNA fragment analysis described in this paper. In these studies, relative transcript expressions are calculated compared with the expression levels of the corresponding transcripts detected in NDs. (B) This figure is a summary of coculture experiments performed 3 times. HEK293T cells expressing NOTCH2-FL-GFP, NOTCH2-Va-GFP, and NOTCH2-Vb-GFP were cocultured 24 hours with 3T3 cells (−Jagged2) or 3T3 cells expressing Jagged2 (+Jagged2). After incubation, cells were scraped, and GFP-positive cells were sorted. Western blotting analysis for Notch2 and Hes 1 was performed as described in “Materials and methods”. Western blots were quantified using the ImageJ software (http://rsb.info.nih.gov/ij). Densitometry measurements were normalized to loading control amount. (C) FLT3-FL, FLT3-Va, and FLT3Vb splice variants were stably expressed in HEK293T cells. Serum-deprived, transfected cells were stimulated for 10 minutes with FLT3L. Cellular tyrosine phosphorylation was analyzed by immunoblotting with a pTyr antibody (Clone 4G10 from Millipore). Elevation of tyrosine phosphorylation of bands at approximately 100 kDa was determined compared with FLT3L unstimulated cells. Additionally, in the samples, Flt3 expression was determined by immunoblotting using anti-FLT3 antibodies from eBiosciences. As a loading control, the same membranes were reprobed with anti-actin antibody. As a control, MOLM14 and HEK293T-GFP stimulated and unstimulated cell lysates were used for pTyr immunoblotting analyses. (D) Serum-starved blasts from patients were stimulated for 10 minutes with 100 ng/mL Flt3L and washed, and cell lysates were prepared to measure phosphorylation levels of STAT5, Akt, and Erk using InstantOne enzyme-linked immunosorbent assay kits according to the manufacturer’s suggestions. Absorbance was measured at 450 nm using an automated enzyme-linked immunosorbent assay plate reader. In the figure, results are presented as bar graphs. The x-axis shows the samples analyzed, and the y-axis displays the phosphorylation level as an absorbance. Results obtained from positive and negative control samples are not displayed on the graph. Expression levels of FLT3-FL and its splice variants in patient samples are reported in a table included in this figure and presented as RFU = log2RFU (relative fluorescence units, described in Figure 3).

Functional effects of NOTCH2 and FLT3 splice variants. (A) This figure displays unsupervised clustering analyses results obtained from the TaqMan gene expression assays of Notch2 target genes HES1, DXT1 and HEY1 carried out in 49 AML patients expressing NOTCH2-FL, FLT3-FL, and their splice variants. Expression levels of NOTCH2-FL, FLT3-FL, and their splice variants were determined by RT-PCR and DNA fragment analysis described in this paper. In these studies, relative transcript expressions are calculated compared with the expression levels of the corresponding transcripts detected in NDs. (B) This figure is a summary of coculture experiments performed 3 times. HEK293T cells expressing NOTCH2-FL-GFP, NOTCH2-Va-GFP, and NOTCH2-Vb-GFP were cocultured 24 hours with 3T3 cells (−Jagged2) or 3T3 cells expressing Jagged2 (+Jagged2). After incubation, cells were scraped, and GFP-positive cells were sorted. Western blotting analysis for Notch2 and Hes 1 was performed as described in “Materials and methods”. Western blots were quantified using the ImageJ software (http://rsb.info.nih.gov/ij). Densitometry measurements were normalized to loading control amount. (C) FLT3-FL, FLT3-Va, and FLT3Vb splice variants were stably expressed in HEK293T cells. Serum-deprived, transfected cells were stimulated for 10 minutes with FLT3L. Cellular tyrosine phosphorylation was analyzed by immunoblotting with a pTyr antibody (Clone 4G10 from Millipore). Elevation of tyrosine phosphorylation of bands at approximately 100 kDa was determined compared with FLT3L unstimulated cells. Additionally, in the samples, Flt3 expression was determined by immunoblotting using anti-FLT3 antibodies from eBiosciences. As a loading control, the same membranes were reprobed with anti-actin antibody. As a control, MOLM14 and HEK293T-GFP stimulated and unstimulated cell lysates were used for pTyr immunoblotting analyses. (D) Serum-starved blasts from patients were stimulated for 10 minutes with 100 ng/mL Flt3L and washed, and cell lysates were prepared to measure phosphorylation levels of STAT5, Akt, and Erk using InstantOne enzyme-linked immunosorbent assay kits according to the manufacturer’s suggestions. Absorbance was measured at 450 nm using an automated enzyme-linked immunosorbent assay plate reader. In the figure, results are presented as bar graphs. The x-axis shows the samples analyzed, and the y-axis displays the phosphorylation level as an absorbance. Results obtained from positive and negative control samples are not displayed on the graph. Expression levels of FLT3-FL and its splice variants in patient samples are reported in a table included in this figure and presented as RFU = log2RFU (relative fluorescence units, described in Figure 3).

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