Inhibition of the interaction between GATA-1 and PU.1 by FOG-1 and PU.1ΔC. (A) Immunoprecipitation assays. 293T cells were cotransfected with various combinations of GATA-1, FOG-1, PU.1, and PU.1ΔC as shown. Immunoprecipitation was carried out using anti-GATA-1 antibodies. (B) Reporter assays using TK-100-PU× 3-Luc, which contains 3 synthetic ETS recognition sites. CV-1 cells were cotransfected with various combinations of GATA-1, FOG-1, PU.1, and PU.1ΔC as shown and with the PU.1-responsible reporter construct and the RL-TK control reporter. The relative activities, normalized by RL-TK activity, are shown. Error bars represent SEM. (C) Structure of PU.1 and PU.1ΔC. (D) May-Giemsa staining of day 14-induced cells infected with MY-IRES-EGFP (EGFP) and MY-PU.1ΔC-IRES-EGFP (PU.1ΔC) on day 8. The EGFP⁺ cells were sorted. Bar represents 50 μm. (E) FACS analysis of day 14-induced EGFP⁺ cells stained with anti-c-KIT and GR-1 antibodies. Antibody staining (blue line) and IgG control staining (gray line) were shown. (F) Percentage of each type of colony. Day-8 induced cells were infected with MY-IRES-EGFP and MY-PU.1ΔC-IRES-EGFP, and the EGFP⁺ cells were sorted on day 10. The sorted cells were transferred to methylcellulose media containing IL-3. Single colonies were selected and analyzed after 7 days of culture.