Figure 2
Figure 2. Validation of interaction between GATA1 and partner proteins in L8057 cells and primary megakaryocytes. (A) Nuclear extracts from BirA biotin ligase expressing (BirA) and BirA and biotag GATA1 expressing (BirA/biotag GATA1) cells were studied. In indicates input lane (20 μg crude nuclear extract); PD, pull-down lane (nuclear extracts precipitated with streptavidin beads); and Un, unbound supernatant lane (proteins not bound to streptavidin). The antibodies used in the Western blot analysis are indicated on the right of the panels. Vertical line(s) have been inserted to indicate a repositioned gel lane. Biotag GATA1 is pulled down from BirA/bioGATA1-transfected cells but is absent from BirA-only cells (top panel). The positions of endogenous (bottom band) and biotag GATA1 (top band) are indicated. (B) Nuclear extracts from untransfected L8057 cells were immunoprecipitated (IP) with αGATA1 antibody. In indicates 20 μg crude nuclear extract; IP, proteins immunoprecipitated by αGATA1 antibody; Un, unbound proteins left in the supernatant after immunoprecipitation; and IgG, control was immunoprecipitation performed with the corresponding normal IgG. Antibodies used in Western blot analysis are indicated on the right of the panels. FOG1, all members of the pentameric complex, several members of the NuRD complex, as well as ETO2, GFI1B, and ZFP143 all coimmunoprecipitate with GATA1. (C-E) Reverse coimmunoprecipitation experiments. L8057 cell nuclear extracts were immunoprecipitated with antibodies against ETO2 (C), GFI1B (D), or ZFP143 (E). Lanes are marked as in panel A. Antibodies used in Western blot analysis are marked on the right of the panels. (F) Mouse bone marrow cells were cultured for 3 days with thrombopoietin. Percentage of CD41 expressing primary megakaryocytes was assessed by fluorescence-activated cell sorter analysis (left). May-Grunwald-Giemsa staining (right) shows that the morphology of the cells used for immunoprecipitation experiments were a mixture of immature and mature megakaryocytes. (G) Nuclear extracts prepared from primary megakaryocytes shown in panel F were immunoprecipitated with αGATA1 antibody. Coimmunoprecipitated proteins were detected by Western blot analysis. Antibodies used in the Western blot analysis are indicated on the right of the panels. (H) Analysis of Eto2 mRNA expression during megakaryocyte maturation. Mouse bone marrow cells were cultured with thrombopoietin for 3 days. CD41 expressing cells were isolated on each day of culture. May-Grunwald-Giemsa staining shows the morphology of the cells isolated on each day (top panel). All panels were photographed at 40× magnification. These cells were used to extract RNA and make cDNA. Eto2 mRNA levels were quantitated relative to GAPDH levels by Taqman real-time RT-PCR, at each time point (bottom panel).

Validation of interaction between GATA1 and partner proteins in L8057 cells and primary megakaryocytes. (A) Nuclear extracts from BirA biotin ligase expressing (BirA) and BirA and biotag GATA1 expressing (BirA/biotag GATA1) cells were studied. In indicates input lane (20 μg crude nuclear extract); PD, pull-down lane (nuclear extracts precipitated with streptavidin beads); and Un, unbound supernatant lane (proteins not bound to streptavidin). The antibodies used in the Western blot analysis are indicated on the right of the panels. Vertical line(s) have been inserted to indicate a repositioned gel lane. Biotag GATA1 is pulled down from BirA/bioGATA1-transfected cells but is absent from BirA-only cells (top panel). The positions of endogenous (bottom band) and biotag GATA1 (top band) are indicated. (B) Nuclear extracts from untransfected L8057 cells were immunoprecipitated (IP) with αGATA1 antibody. In indicates 20 μg crude nuclear extract; IP, proteins immunoprecipitated by αGATA1 antibody; Un, unbound proteins left in the supernatant after immunoprecipitation; and IgG, control was immunoprecipitation performed with the corresponding normal IgG. Antibodies used in Western blot analysis are indicated on the right of the panels. FOG1, all members of the pentameric complex, several members of the NuRD complex, as well as ETO2, GFI1B, and ZFP143 all coimmunoprecipitate with GATA1. (C-E) Reverse coimmunoprecipitation experiments. L8057 cell nuclear extracts were immunoprecipitated with antibodies against ETO2 (C), GFI1B (D), or ZFP143 (E). Lanes are marked as in panel A. Antibodies used in Western blot analysis are marked on the right of the panels. (F) Mouse bone marrow cells were cultured for 3 days with thrombopoietin. Percentage of CD41 expressing primary megakaryocytes was assessed by fluorescence-activated cell sorter analysis (left). May-Grunwald-Giemsa staining (right) shows that the morphology of the cells used for immunoprecipitation experiments were a mixture of immature and mature megakaryocytes. (G) Nuclear extracts prepared from primary megakaryocytes shown in panel F were immunoprecipitated with αGATA1 antibody. Coimmunoprecipitated proteins were detected by Western blot analysis. Antibodies used in the Western blot analysis are indicated on the right of the panels. (H) Analysis of Eto2 mRNA expression during megakaryocyte maturation. Mouse bone marrow cells were cultured with thrombopoietin for 3 days. CD41 expressing cells were isolated on each day of culture. May-Grunwald-Giemsa staining shows the morphology of the cells isolated on each day (top panel). All panels were photographed at 40× magnification. These cells were used to extract RNA and make cDNA. Eto2 mRNA levels were quantitated relative to GAPDH levels by Taqman real-time RT-PCR, at each time point (bottom panel).

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