E2f4 is highly expressed in fetal liver erythroid cells. (A) E2f4 protein expression by immunohistochemistry in E12.5 E2f4^+/+ (left panels) and E2f4^–/– (right panels) embryo sections, counterstained with hematoxylin (blue). High-power images of the FL are shown in the bottom panels. Lack of detectable signal in the E2f4^–/– littermate control demonstrates the specificity of the E2f4 antibody used in these studies. (B) Sorting protocol to isolate erythroid differentiation populations from E15.5 E2f4^+/+ FL. Mature erythroblasts (Ter119^hi/c-kit^neg, M), proerythroblasts (Ter119^dim/c-kit^dim, P), and hematopoietic progenitors (Ter119^neg/c-kit^hi, H; include the earliest erythroid progenitors, BFU-Es and CFU-Es). Morphology (benzidine and May-Grunwald-Giemsa stain) and cell cycle profiles by PI were used to characterize these populations. (C) Protein expression of E2F and pRB family members in sorted E15.5 E2f4^+/+ FL subpopulations was assessed by Western blot; mSin3A indicates loading control. (D) Gel-shift analyses of E2F/pRb family complexes in E12.5 E2f4^+/+ andE2f4^–/– FL. (E) Gel-shift analysis of E2F/pRb family complexes in sorted E15.5 E2f4^+/+ FL populations. Protein samples were derived from whole-cell extracts except where denoted. Nuc indicates nuclear extracts; WCE, whole-cell extracts. Solid arrowheads denote complexes supershifted by E2F4 polyclonal antibody, and clear arrowhead (Dii) denotes supershift with p107 mAb (SD6; a kind gift from N. Dyson), whereas an alternate p107 pAb (SC318X; Santa Cruz, Santa Cruz, CA) results in destruction of the complex (Diii,E).