Figure 5.
Figure 5. The interaction between ZNF423α and EBF-1 inhibits EBF-1 target gene expression in hematopoietic cells. (A) Co-immunoprecipitation of ZNF423 and EBF-1. Immunoblotting of input and eluate from Flag-IP. Detection of the transfected proteins by indicated antibodies. Binding was shown in three independent experiments. (B) Transactivation of the CD79b promoter upon EBF-1 and ZNF423α wild-type versus C-terminal deletion mutant ZNF423αΔ28-30. Luciferase assay was performed in 293T cells. ZNF423α plasmid was co-transfected at the indicated concentrations. Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars represent SD of three technical replicates. Significance is calculated by Student’s t test, comparing indicated samples (***, P ≤ 0.001). Data were reproduced in three independent experiments. (C) ZNF423α concentration-dependent transactivation of the CD79b promoter. Luciferase assay was performed as in B. Significance of technical replicates is calculated by Student’s t test, comparing to CD79b promoter baseline (***, P ≤ 0.001). Data were reproduced in three independent experiments. (D) ZNF423 domain mapping using deletion mutants. Each box represents a C2H2 zinc finger motif. DBD, (light gray box) DNA binding; PBD (black box), protein binding; dark gray box, unknown function; black lines, deleted regions. Scheme is not to scale. (E) CD79b promoter activity in dependence of EBF-1 and co-transfected ZNF423 isoforms and mutants. Luciferase assay was performed as in B. Significance of technical replicates is calculated by Student’s t test, comparing to CD79b promoter activities in the presence of EBF-1 alone to cotransfected ZNF423 isoforms or mutants(**, P ≤ 0.01; ***, P ≤ 0.001; n.s., not significant). Data were reproduced in three independent experiments. (F) Expression of recombinant ZNF423 mutants. Immunoblotting of 293T cell lysates after transfection with Flag-ZNF423 mutants. Detection of the transfected proteins by anti-Flag antibody. (G) Expression of EBF-1 and its target genes upon retrovirally forced expression of ZNF423α in CD34+ stem/progenitor cells. CD34+ were transduced via retroviral particles and cultured for indicated days. Relative fold change was measured by qPCR (2−ΔΔCt). Transcript levels were normalized to B2M and pMYs-control cells. Error bars represent SD of mean fold induction from independent experiments (day 5, n = 6; day 8, n = 4). Significance is calculated using 2−ΔCt values by Student’s t test (*, P ≤ 0.05).

The interaction between ZNF423α and EBF-1 inhibits EBF-1 target gene expression in hematopoietic cells. (A) Co-immunoprecipitation of ZNF423 and EBF-1. Immunoblotting of input and eluate from Flag-IP. Detection of the transfected proteins by indicated antibodies. Binding was shown in three independent experiments. (B) Transactivation of the CD79b promoter upon EBF-1 and ZNF423α wild-type versus C-terminal deletion mutant ZNF423αΔ28-30. Luciferase assay was performed in 293T cells. ZNF423α plasmid was co-transfected at the indicated concentrations. Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars represent SD of three technical replicates. Significance is calculated by Student’s t test, comparing indicated samples (***, P ≤ 0.001). Data were reproduced in three independent experiments. (C) ZNF423α concentration-dependent transactivation of the CD79b promoter. Luciferase assay was performed as in B. Significance of technical replicates is calculated by Student’s t test, comparing to CD79b promoter baseline (***, P ≤ 0.001). Data were reproduced in three independent experiments. (D) ZNF423 domain mapping using deletion mutants. Each box represents a C2H2 zinc finger motif. DBD, (light gray box) DNA binding; PBD (black box), protein binding; dark gray box, unknown function; black lines, deleted regions. Scheme is not to scale. (E) CD79b promoter activity in dependence of EBF-1 and co-transfected ZNF423 isoforms and mutants. Luciferase assay was performed as in B. Significance of technical replicates is calculated by Student’s t test, comparing to CD79b promoter activities in the presence of EBF-1 alone to cotransfected ZNF423 isoforms or mutants(**, P ≤ 0.01; ***, P ≤ 0.001; n.s., not significant). Data were reproduced in three independent experiments. (F) Expression of recombinant ZNF423 mutants. Immunoblotting of 293T cell lysates after transfection with Flag-ZNF423 mutants. Detection of the transfected proteins by anti-Flag antibody. (G) Expression of EBF-1 and its target genes upon retrovirally forced expression of ZNF423α in CD34+ stem/progenitor cells. CD34+ were transduced via retroviral particles and cultured for indicated days. Relative fold change was measured by qPCR (2−ΔΔCt). Transcript levels were normalized to B2M and pMYs-control cells. Error bars represent SD of mean fold induction from independent experiments (day 5, n = 6; day 8, n = 4). Significance is calculated using 2−ΔCt values by Student’s t test (*, P ≤ 0.05).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal