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A , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on FLT3 + and FLT3 − HSPCs treated with 50 ng/ml IL-4 in vitro for 30 minutes (n=3; FMO, fluorescence minus one; MFI, mean fluorescence intensity). B , Immunoblot analysis of 293T cells co-expressing Stat6 -HA and Il4ra -Myc with GFP -Flag, Flt3 (Full Length)-Flag, Flt3 (ΔKinase)-Flag, or Flt3 -Flag plus inhibitor (Quizartinib: 20nM) for 24 h. C , Immunoblot analysis of 293T cells co-expressing JAK1 , Il4ra or Stat6 with GFP -Flag or Flt3 (Full Length)-Flag for 24 h. D , Immunoblot analysis of 293T cells co-expressing Flt3 -Flag, Stat6 -HA and Il4ra -Myc for 24 h and then co-IP were performed <t>with</t> <t>anti-Flag</t> antibody (IP, immunoprecipitation, WCL, whole-cell lysate). E , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on HSPCs treated with 100 ng/ml FLT3L in vitro for 30 minutes (n=3). F , Immunoblot analysis of 293T cells co-expressing human STAT6 -HA and IL4R -Myc with GFP -Flag, FLT3 (WT)-Flag or FLT3 (ITD)-Flag for 24 h. G , Immunoblot analysis of HL-60 and MOLM-13 cells treated with human IL-4 and/or FLT3 inhibitor (Quizartinib: 20 nM) for 1 hours. H , Representative FACS plots and quantification of the CD19 + B220 + B lymphocytes differentiated from HSPCs at day 10 on OP9 stromal cells supplemented with FLT3L and IL-7 (n=4). I , Percentage of MPP4 within isolated HSPCs, and the MPP_Lym/Mye ratio, with or without pre-treatment using FLT3 inhibitor (Quizartinib: 20 nM) followed by 50 ng/ml IL-4 for 24 hours in vitro (n=3). All data represent means ± s.e.m. Statistical significance was determined by unpaired two-tailed Student’s t-test ( A , E, H and I ).
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A , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on FLT3 + and FLT3 − HSPCs treated with 50 ng/ml IL-4 in vitro for 30 minutes (n=3; FMO, fluorescence minus one; MFI, mean fluorescence intensity). B , Immunoblot analysis of 293T cells co-expressing Stat6 -HA and Il4ra -Myc with GFP -Flag, Flt3 (Full Length)-Flag, Flt3 (ΔKinase)-Flag, or Flt3 -Flag plus inhibitor (Quizartinib: 20nM) for 24 h. C , Immunoblot analysis of 293T cells co-expressing JAK1 , Il4ra or Stat6 with GFP -Flag or Flt3 (Full Length)-Flag for 24 h. D , Immunoblot analysis of 293T cells co-expressing Flt3 -Flag, Stat6 -HA and Il4ra -Myc for 24 h and then co-IP were performed <t>with</t> <t>anti-Flag</t> antibody (IP, immunoprecipitation, WCL, whole-cell lysate). E , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on HSPCs treated with 100 ng/ml FLT3L in vitro for 30 minutes (n=3). F , Immunoblot analysis of 293T cells co-expressing human STAT6 -HA and IL4R -Myc with GFP -Flag, FLT3 (WT)-Flag or FLT3 (ITD)-Flag for 24 h. G , Immunoblot analysis of HL-60 and MOLM-13 cells treated with human IL-4 and/or FLT3 inhibitor (Quizartinib: 20 nM) for 1 hours. H , Representative FACS plots and quantification of the CD19 + B220 + B lymphocytes differentiated from HSPCs at day 10 on OP9 stromal cells supplemented with FLT3L and IL-7 (n=4). I , Percentage of MPP4 within isolated HSPCs, and the MPP_Lym/Mye ratio, with or without pre-treatment using FLT3 inhibitor (Quizartinib: 20 nM) followed by 50 ng/ml IL-4 for 24 hours in vitro (n=3). All data represent means ± s.e.m. Statistical significance was determined by unpaired two-tailed Student’s t-test ( A , E, H and I ).
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A , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on FLT3 + and FLT3 − HSPCs treated with 50 ng/ml IL-4 in vitro for 30 minutes (n=3; FMO, fluorescence minus one; MFI, mean fluorescence intensity). B , Immunoblot analysis of 293T cells co-expressing Stat6 -HA and Il4ra -Myc with GFP -Flag, Flt3 (Full Length)-Flag, Flt3 (ΔKinase)-Flag, or Flt3 -Flag plus inhibitor (Quizartinib: 20nM) for 24 h. C , Immunoblot analysis of 293T cells co-expressing JAK1 , Il4ra or Stat6 with GFP -Flag or Flt3 (Full Length)-Flag for 24 h. D , Immunoblot analysis of 293T cells co-expressing Flt3 -Flag, Stat6 -HA and Il4ra -Myc for 24 h and then co-IP were performed <t>with</t> <t>anti-Flag</t> antibody (IP, immunoprecipitation, WCL, whole-cell lysate). E , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on HSPCs treated with 100 ng/ml FLT3L in vitro for 30 minutes (n=3). F , Immunoblot analysis of 293T cells co-expressing human STAT6 -HA and IL4R -Myc with GFP -Flag, FLT3 (WT)-Flag or FLT3 (ITD)-Flag for 24 h. G , Immunoblot analysis of HL-60 and MOLM-13 cells treated with human IL-4 and/or FLT3 inhibitor (Quizartinib: 20 nM) for 1 hours. H , Representative FACS plots and quantification of the CD19 + B220 + B lymphocytes differentiated from HSPCs at day 10 on OP9 stromal cells supplemented with FLT3L and IL-7 (n=4). I , Percentage of MPP4 within isolated HSPCs, and the MPP_Lym/Mye ratio, with or without pre-treatment using FLT3 inhibitor (Quizartinib: 20 nM) followed by 50 ng/ml IL-4 for 24 hours in vitro (n=3). All data represent means ± s.e.m. Statistical significance was determined by unpaired two-tailed Student’s t-test ( A , E, H and I ).
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A , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on FLT3 + and FLT3 − HSPCs treated with 50 ng/ml IL-4 in vitro for 30 minutes (n=3; FMO, fluorescence minus one; MFI, mean fluorescence intensity). B , Immunoblot analysis of 293T cells co-expressing Stat6 -HA and Il4ra -Myc with GFP -Flag, Flt3 (Full Length)-Flag, Flt3 (ΔKinase)-Flag, or Flt3 -Flag plus inhibitor (Quizartinib: 20nM) for 24 h. C , Immunoblot analysis of 293T cells co-expressing JAK1 , Il4ra or Stat6 with GFP -Flag or Flt3 (Full Length)-Flag for 24 h. D , Immunoblot analysis of 293T cells co-expressing Flt3 -Flag, Stat6 -HA and Il4ra -Myc for 24 h and then co-IP were performed <t>with</t> <t>anti-Flag</t> antibody (IP, immunoprecipitation, WCL, whole-cell lysate). E , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on HSPCs treated with 100 ng/ml FLT3L in vitro for 30 minutes (n=3). F , Immunoblot analysis of 293T cells co-expressing human STAT6 -HA and IL4R -Myc with GFP -Flag, FLT3 (WT)-Flag or FLT3 (ITD)-Flag for 24 h. G , Immunoblot analysis of HL-60 and MOLM-13 cells treated with human IL-4 and/or FLT3 inhibitor (Quizartinib: 20 nM) for 1 hours. H , Representative FACS plots and quantification of the CD19 + B220 + B lymphocytes differentiated from HSPCs at day 10 on OP9 stromal cells supplemented with FLT3L and IL-7 (n=4). I , Percentage of MPP4 within isolated HSPCs, and the MPP_Lym/Mye ratio, with or without pre-treatment using FLT3 inhibitor (Quizartinib: 20 nM) followed by 50 ng/ml IL-4 for 24 hours in vitro (n=3). All data represent means ± s.e.m. Statistical significance was determined by unpaired two-tailed Student’s t-test ( A , E, H and I ).
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A , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on FLT3 + and FLT3 − HSPCs treated with 50 ng/ml IL-4 in vitro for 30 minutes (n=3; FMO, fluorescence minus one; MFI, mean fluorescence intensity). B , Immunoblot analysis of 293T cells co-expressing Stat6 -HA and Il4ra -Myc with GFP -Flag, Flt3 (Full Length)-Flag, Flt3 (ΔKinase)-Flag, or Flt3 -Flag plus inhibitor (Quizartinib: 20nM) for 24 h. C , Immunoblot analysis of 293T cells co-expressing JAK1 , Il4ra or Stat6 with GFP -Flag or Flt3 (Full Length)-Flag for 24 h. D , Immunoblot analysis of 293T cells co-expressing Flt3 -Flag, Stat6 -HA and Il4ra -Myc for 24 h and then co-IP were performed <t>with</t> <t>anti-Flag</t> antibody (IP, immunoprecipitation, WCL, whole-cell lysate). E , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on HSPCs treated with 100 ng/ml FLT3L in vitro for 30 minutes (n=3). F , Immunoblot analysis of 293T cells co-expressing human STAT6 -HA and IL4R -Myc with GFP -Flag, FLT3 (WT)-Flag or FLT3 (ITD)-Flag for 24 h. G , Immunoblot analysis of HL-60 and MOLM-13 cells treated with human IL-4 and/or FLT3 inhibitor (Quizartinib: 20 nM) for 1 hours. H , Representative FACS plots and quantification of the CD19 + B220 + B lymphocytes differentiated from HSPCs at day 10 on OP9 stromal cells supplemented with FLT3L and IL-7 (n=4). I , Percentage of MPP4 within isolated HSPCs, and the MPP_Lym/Mye ratio, with or without pre-treatment using FLT3 inhibitor (Quizartinib: 20 nM) followed by 50 ng/ml IL-4 for 24 hours in vitro (n=3). All data represent means ± s.e.m. Statistical significance was determined by unpaired two-tailed Student’s t-test ( A , E, H and I ).
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A , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on FLT3 + and FLT3 − HSPCs treated with 50 ng/ml IL-4 in vitro for 30 minutes (n=3; FMO, fluorescence minus one; MFI, mean fluorescence intensity). B , Immunoblot analysis of 293T cells co-expressing Stat6 -HA and Il4ra -Myc with GFP -Flag, Flt3 (Full Length)-Flag, Flt3 (ΔKinase)-Flag, or Flt3 -Flag plus inhibitor (Quizartinib: 20nM) for 24 h. C , Immunoblot analysis of 293T cells co-expressing JAK1 , Il4ra or Stat6 with GFP -Flag or Flt3 (Full Length)-Flag for 24 h. D , Immunoblot analysis of 293T cells co-expressing Flt3 -Flag, Stat6 -HA and Il4ra -Myc for 24 h and then co-IP were performed <t>with</t> <t>anti-Flag</t> antibody (IP, immunoprecipitation, WCL, whole-cell lysate). E , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on HSPCs treated with 100 ng/ml FLT3L in vitro for 30 minutes (n=3). F , Immunoblot analysis of 293T cells co-expressing human STAT6 -HA and IL4R -Myc with GFP -Flag, FLT3 (WT)-Flag or FLT3 (ITD)-Flag for 24 h. G , Immunoblot analysis of HL-60 and MOLM-13 cells treated with human IL-4 and/or FLT3 inhibitor (Quizartinib: 20 nM) for 1 hours. H , Representative FACS plots and quantification of the CD19 + B220 + B lymphocytes differentiated from HSPCs at day 10 on OP9 stromal cells supplemented with FLT3L and IL-7 (n=4). I , Percentage of MPP4 within isolated HSPCs, and the MPP_Lym/Mye ratio, with or without pre-treatment using FLT3 inhibitor (Quizartinib: 20 nM) followed by 50 ng/ml IL-4 for 24 hours in vitro (n=3). All data represent means ± s.e.m. Statistical significance was determined by unpaired two-tailed Student’s t-test ( A , E, H and I ).
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A , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on FLT3 + and FLT3 − HSPCs treated with 50 ng/ml IL-4 in vitro for 30 minutes (n=3; FMO, fluorescence minus one; MFI, mean fluorescence intensity). B , Immunoblot analysis of 293T cells co-expressing Stat6 -HA and Il4ra -Myc with GFP -Flag, Flt3 (Full Length)-Flag, Flt3 (ΔKinase)-Flag, or Flt3 -Flag plus inhibitor (Quizartinib: 20nM) for 24 h. C , Immunoblot analysis of 293T cells co-expressing JAK1 , Il4ra or Stat6 with GFP -Flag or Flt3 (Full Length)-Flag for 24 h. D , Immunoblot analysis of 293T cells co-expressing Flt3 -Flag, Stat6 -HA and Il4ra -Myc for 24 h and then co-IP were performed with anti-Flag antibody (IP, immunoprecipitation, WCL, whole-cell lysate). E , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on HSPCs treated with 100 ng/ml FLT3L in vitro for 30 minutes (n=3). F , Immunoblot analysis of 293T cells co-expressing human STAT6 -HA and IL4R -Myc with GFP -Flag, FLT3 (WT)-Flag or FLT3 (ITD)-Flag for 24 h. G , Immunoblot analysis of HL-60 and MOLM-13 cells treated with human IL-4 and/or FLT3 inhibitor (Quizartinib: 20 nM) for 1 hours. H , Representative FACS plots and quantification of the CD19 + B220 + B lymphocytes differentiated from HSPCs at day 10 on OP9 stromal cells supplemented with FLT3L and IL-7 (n=4). I , Percentage of MPP4 within isolated HSPCs, and the MPP_Lym/Mye ratio, with or without pre-treatment using FLT3 inhibitor (Quizartinib: 20 nM) followed by 50 ng/ml IL-4 for 24 hours in vitro (n=3). All data represent means ± s.e.m. Statistical significance was determined by unpaired two-tailed Student’s t-test ( A , E, H and I ).

Journal: Immunity

Article Title: Activating an Interleukin-4-FLT3-STAT6 axis in Multipotent Progenitors Restores Lymphopoiesis in Inflammation and Aging

doi: 10.1016/j.immuni.2026.04.018

Figure Lengend Snippet: A , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on FLT3 + and FLT3 − HSPCs treated with 50 ng/ml IL-4 in vitro for 30 minutes (n=3; FMO, fluorescence minus one; MFI, mean fluorescence intensity). B , Immunoblot analysis of 293T cells co-expressing Stat6 -HA and Il4ra -Myc with GFP -Flag, Flt3 (Full Length)-Flag, Flt3 (ΔKinase)-Flag, or Flt3 -Flag plus inhibitor (Quizartinib: 20nM) for 24 h. C , Immunoblot analysis of 293T cells co-expressing JAK1 , Il4ra or Stat6 with GFP -Flag or Flt3 (Full Length)-Flag for 24 h. D , Immunoblot analysis of 293T cells co-expressing Flt3 -Flag, Stat6 -HA and Il4ra -Myc for 24 h and then co-IP were performed with anti-Flag antibody (IP, immunoprecipitation, WCL, whole-cell lysate). E , Representative FACS plots and quantification of the intercellular staining of pSTAT6 on HSPCs treated with 100 ng/ml FLT3L in vitro for 30 minutes (n=3). F , Immunoblot analysis of 293T cells co-expressing human STAT6 -HA and IL4R -Myc with GFP -Flag, FLT3 (WT)-Flag or FLT3 (ITD)-Flag for 24 h. G , Immunoblot analysis of HL-60 and MOLM-13 cells treated with human IL-4 and/or FLT3 inhibitor (Quizartinib: 20 nM) for 1 hours. H , Representative FACS plots and quantification of the CD19 + B220 + B lymphocytes differentiated from HSPCs at day 10 on OP9 stromal cells supplemented with FLT3L and IL-7 (n=4). I , Percentage of MPP4 within isolated HSPCs, and the MPP_Lym/Mye ratio, with or without pre-treatment using FLT3 inhibitor (Quizartinib: 20 nM) followed by 50 ng/ml IL-4 for 24 hours in vitro (n=3). All data represent means ± s.e.m. Statistical significance was determined by unpaired two-tailed Student’s t-test ( A , E, H and I ).

Article Snippet: The supernatant was incubated with Anti-Flag Magnetic Beads (MedChem Express) at 4 °C overnight.

Techniques: Staining, In Vitro, Fluorescence, Western Blot, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, Isolation, Two Tailed Test