Arf induction in prelymphomatous Eμ-Myc mice. (A) B220⁺ cells were purified from the bone marrow (BM) and spleens (Sp) of nontransgenic control C57BL/6 (Con) mice or from syngeneic animals expressing the Eμ-Myc (M) transgene. Cells were harvested from healthy mice at biweekly (w) intervals after birth. Cell lysates (25 μg protein per lane from B220⁺ cells purified from BM or Sp) were separated on denaturing gels, and proteins were immunoblotted with antibodies directed to p19^Arf (5-C3-1),¹⁰  c-Myc (N-262; Santa Cruz Biotechnology, Santa Cruz, CA), p53 (NCL-p53-505; Novocastra, Newcastle upon Tyne, United Kingdom), p21 (F5; Santa Cruz), or beta-actin (A-5441; Sigma, St Louis, MO), as indicated at the left of the panels. Cultured Arf-null B cells were untreated (lane 1) or irradiated with 5 Gy 2 hours prior to lysis (lane 2) to induce higher levels of p53. Cultured p53-null B cells express very high levels of p19^Arf (lane 3). (B) Lysates (in micrograms of total protein as indicated at the bottom of the panel) of B220⁺ BM cells and splenocytes (Sp) purified from control (Con) or Eμ-Myc (M) transgenic mice were immunoblotted with antibodies to p19^Arf. The signals were compared with those generated with various quantities of lysate protein obtained from a representative p53-null lymphoma and from cultured p53-null B cells. (C) Cultured B cells from p53-null mice were stained with an isotype-matched control mAb (panel Ci, negative control) or with mAb 5-C3-1 to p19^Arf (panel Cii; p53-null cells as positive control) conjugated to AlexaFluor-647. Arf-null cells served as an additional negative control for mAb 5-C3-1 staining (Ciii). BM and Sp cells from control [Myc (−)] or transgenic [Myc (+)] mice of 2 weeks of age were assayed by dual-color FC using antibodies to B220 and to p19^Arf (panels Civ-Cix). Gated B220⁺ cells in BM (Civ-Cvi) and Sp cells (Cvii-Cix) were analyzed for reactivity with AlexaFluor-647–conjugated mAb 5-C3-1 directed to p19^Arf. Vertical dashed lines indicate fluorescence intensities in negative control panels (Ci, Civ, Cviii).