Figure 2. Biosynthesis and Western blotting of proHNP and HNP in HNP-1 mice deficient in C/EBP-ε.

Murine bone marrow (BM) cells from one wild type and transgenic HNP-1 mice with or without deficiency in C/EBP-ε were extracted. (A) Cells were pulsed with ³⁵S-methionine/cysteine for 1 hour and chased for 5 hours. Cell lysates and medium were immunoprecipitated with antibodies against proHNP and HNP. Immunoprecipitates were pooled and analyzed by 16% SDS-Tricine-PAGE and fluorography. (B) Non-granulocytic cells from BM of wild type mice, transgenic HNP-1 mice (HNP-1, Cebpe^+/+), and HNP-1 mice heterozygous or deficient for C/EBP-ε (HNP-1, Cebpe^+/− or Cebpe^-/-) were removed by ammonium chloride-based lysis and by immunomagnetic sorting. Western blotting was performed for HNP, proHNP, and GAPDH and is representative of two independent experiments. (C) BM cells from Cebpe^+/+ (blue) or Cebpe^-/-mice (orange) were immunomagnetically depleted of non-granulocytic cells and labeled with biotinylated rat anti-mouse CD11b antibody and analyzed by flow cytometry using biotinylated rat anti-mouse IgG2b as isotype control. Data are representative of four independent experiments.