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. 2010 Jun 7;207(6):1183–1195. doi: 10.1084/jem.20092215

Figure 4.

Figure 4.

Decreased GATA1 activity and GATA1-responsive genes in SENP1 KO fetal liver and stage-matched hematopoietic progenitors. (A) Expression of GATA1, GATA2, and EpoR was examined in E13.5 fetal liver of WT and SENP1 KO mice by Western blotting with respective antibodies. The relative protein levels were quantified by taking normalized WT (vs. tubulin) as 1.0. Five other embryos of WT or KO exhibited similar results. (B) GATA1 activity in SENP1 KO fetal liver. Nuclear extracts were isolated from E13.5 WT and SENP1 KO fetal livers (n = 3 each group), and an equal amount of GATA1 proteins from WT and KO were used for the GATA1 DNA binding activity assay by EMSA with a GATA1-specific oligonucleotide probe. A nuclear protein USF2 was used as a loading control. A representative gel from three independent experiments is shown. The relative GATA1 DNA-binding activity and protein levels were quantified by taking WT as 1.0. (C) Representative flow cytometric analysis of erythroid differentiation in vivo. Freshly isolated E13.5 WT or SENP1 KO fetal liver cells were doubly stained with anti–CD71-PE and anti–Ter119-APC. Flow cytometry density plots of all viable fetal liver cells from representative WT and SENP1 KO embryo in the same littermates are shown. Regions R1–R5 define distinct populations of erythroid progenitor cells at different stages of differentiation. Primitive progenitor cells (including mature BFU-Es and CFU-Es) and proerythroblasts are shown in R1, proerythroblasts and early basophilic erythroblasts in R2, early and late basophilic erythroblasts in R3, chromatophilic and orthochromatophilic erythroblasts in R4, and late orthochromatophilic and reticulocytes in R5. Numbers represent the percentage of cells within that region. Two independent experiments are performed. (D) Transcripts for EpoR, Hbb-y, c-Myb, and c-Kit from sorted fetal liver cells from R2 areas were quantified by quantitative RT-PCR and normalized to β-actin. Data are mean ± SEM (n = 5 from each group) from two independent experiments. *, P < 0.01 versus WT group.

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