Id2 interacts with PU.1 and modulates PU.1 and GATA-1 activity. (A) Immunoprecipitation was performed using EML cell lysate with anti-PU.1 antibody and IgG. Id2 presence in the immunocomplex was examined by Western blot analysis. (B) Id2 expression was induced in dose-dependent manner by doxycycline in inducible U2OS-Id2 cells. (C) PU.1-pECE, M-CSFR-Luc, and phRL4.7 were cotransfected into U2OS-Id2 cells. At 24 hours after transfection, Id2 expression was induced by addition of doxycycline. Luciferase activity was examined and normalized to renilla activity. (D) Immunoprecipitation was performed in EML-GFP, EML-Id2-GFP, EML-NS-shRNA, and EML-Id2-shRNA with anti-PU.1 antibody. GATA-1 presence in the immunocomplex was examined by Western blot analysis (top panel). GATA-1 and Id2 levels in EML-GFP, EML-Id2-GFP, EML-NS-shRNA, and EML-Id2-shRNA whole-cell lysates were determined by Western blot analysis (bottom panel). (E) pcDNA-GATA-1, PU.1-pECE, p4.2-Luc, and phRL4.7 were cotransfected into U2OS-Id2 cells. At 24 hours after transfection, Id2 expression was induced by addition of doxycycline. Luciferase activity was examined and normalized to renilla activity. (F) Total RNA was purified from EML-GFP or EML-Id2-GFP, and converted to cDNA. GATA-1, ELKF, EpoR, and alpha-globin expression levels were determined by real-time PCR. (G) Ter119⁺CD71⁺ primary erythroblasts were purified from Id2^+/+ and Id2^−/− mice by multicolor-based sorting techniques. Total RNA was purified from sorted cells and converted to cDNA. GATA-1, ELKF, EpoR, and alpha-globin expression levels were determined by real-time PCR.