Figure 2.
Figure 2. FL-induced responses in WT-Flt3, Y589F-Flt3, and Y599F-Flt3-32D cells. (A) The 32D cells were retrovirally infected with WT-Flt3, Y589F-Flt3, or Y599F-Flt3. Bulk populations obtained after selection in 0.2 g/L G418 were tested for their surface expression of Flt3 isoforms by flow cytometry (i: open curve, anti-Flt3; filled curve, isotype control). Kinase activity of the expressed Flt3 isoform was confirmed by immunoprecipitation of FL-stimulated Flt3 and a subsequent in vitro kinase assay with Flt3 as endogenous substrate (ii) as described in “In vitro kinase activity of Flt3 and Lyn” as well as by immunoprecipitation of activated Flt3 from stimulated cells, followed by probing with a phosphotyrosine antibody and subsequent reprobing with a Flt3 antibody to verify equal loading (iii). (B) Time-dependent autophosphorylation of Flt3 isoforms was examined by immunoprecipitation of Flt3 at the indicated time points upon FL stimulation. After SDS-PAGE and immunoblotting (IB), membranes were probed for phosphotyrosine and Flt3, respectively. The intensity of the bands was quantified by densitometric analysis. The ratio of pFlt3 to Flt3, normalized to time point zero, is depicted for each Flt3 isoform (• indicates WT-Flt3; ▪, Y589F-Flt3; ▴, Y599F-Flt3). (C) Dose-dependent rescue from IL-3 withdrawal in the Flt3-32D transfectants by FL was determined by an MTT assay. Thirty thousand cells/well (96-well, triplicates) were starved from IL-3 in the presence of different concentrations of FL and 2% serum. After 48 hours the MTT dye was added for another 4 hours before absorbance of the converted dye was read at 595/630 nm. OD indicates optical density. Bars indicate fold increase of metabolic activity compared with control cells (no IL-3, no FL). ▪ indicates WT-Flt3; , Y589F-Flt3; and □, Y599F-Flt3. Data from 3 individual experiments were subjected to ANOVA statistical analysis. Error bars indicate standard deviation. *P < .05; **P < .01; ***P < .001. (D) To examine FL-induced Erk phosphorylation, 32D transfectants were starved from cytokines for 4 hours and then stimulated with 100 ng/mL FL for the indicated periods of time. After lysis, total cell lysates were subjected to a Western blot analysis for pErk and total Erk, respectively. All experiments were performed at least 3 times with consistent results.

FL-induced responses in WT-Flt3, Y589F-Flt3, and Y599F-Flt3-32D cells. (A) The 32D cells were retrovirally infected with WT-Flt3, Y589F-Flt3, or Y599F-Flt3. Bulk populations obtained after selection in 0.2 g/L G418 were tested for their surface expression of Flt3 isoforms by flow cytometry (i: open curve, anti-Flt3; filled curve, isotype control). Kinase activity of the expressed Flt3 isoform was confirmed by immunoprecipitation of FL-stimulated Flt3 and a subsequent in vitro kinase assay with Flt3 as endogenous substrate (ii) as described in “In vitro kinase activity of Flt3 and Lyn” as well as by immunoprecipitation of activated Flt3 from stimulated cells, followed by probing with a phosphotyrosine antibody and subsequent reprobing with a Flt3 antibody to verify equal loading (iii). (B) Time-dependent autophosphorylation of Flt3 isoforms was examined by immunoprecipitation of Flt3 at the indicated time points upon FL stimulation. After SDS-PAGE and immunoblotting (IB), membranes were probed for phosphotyrosine and Flt3, respectively. The intensity of the bands was quantified by densitometric analysis. The ratio of pFlt3 to Flt3, normalized to time point zero, is depicted for each Flt3 isoform (• indicates WT-Flt3; ▪, Y589F-Flt3; ▴, Y599F-Flt3). (C) Dose-dependent rescue from IL-3 withdrawal in the Flt3-32D transfectants by FL was determined by an MTT assay. Thirty thousand cells/well (96-well, triplicates) were starved from IL-3 in the presence of different concentrations of FL and 2% serum. After 48 hours the MTT dye was added for another 4 hours before absorbance of the converted dye was read at 595/630 nm. OD indicates optical density. Bars indicate fold increase of metabolic activity compared with control cells (no IL-3, no FL). ▪ indicates WT-Flt3; , Y589F-Flt3; and □, Y599F-Flt3. Data from 3 individual experiments were subjected to ANOVA statistical analysis. Error bars indicate standard deviation. *P < .05; **P < .01; ***P < .001. (D) To examine FL-induced Erk phosphorylation, 32D transfectants were starved from cytokines for 4 hours and then stimulated with 100 ng/mL FL for the indicated periods of time. After lysis, total cell lysates were subjected to a Western blot analysis for pErk and total Erk, respectively. All experiments were performed at least 3 times with consistent results.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal