pDP4 is cell type specific. (A) Northern blot with total RNA from various mouse tissues showing that pDP4 is specifically expressed in small intestine and bone marrow (upper panel). The lower panel shows the hybridization of the same membrane with a GAPDH probe as a loading control. (B) pDP4 protein is expressed in bone marrow but not in spleen. Western blot using 60 μg total cell extracts from mouse spleen and bone marrow was incubated with an antiserum directed against the C-terminus of pDP4. Ngase F treatment indicates that pDP4 exists predominately as a glycosylated peptide (glyco-pDP4) in vivo. Multimerized pDP4 (multi-pDP4) can be noted as high molecular fragments. (C) Quantitative real-time RT-PCR of pDP4 transcripts using RNA from defined FACS-sorted hematopoietic cell populations. The following antibodies and mouse organs (in parentheses) were used for the sorting: B220 for B cells (spleen), CD3 for T cells (thymus), F4/80 for macrophages (bone marrow), CD41 for megakaryocytes (bone marrow), Ter119 for erythroid cells (bone marrow), Gr-1/Mac-1 for granulocytes (bone marrow), and a cocktail of all mentioned antibodies for lineage depletion to enrich progenitor cells (bone marrow). pDP4 values of 2 independent experiments are shown as percent expression of 18S rRNA ± standard deviation. (D) Immunofluorescence of a bone marrow cytospin section (original magnification, × 100) costained with anti-Gr-1 (primary)/Alexa-546 red (secondary) and anti-pDP4 (primary)/Alexa-488 green (secondary) antibody combinations as well as DAPI to visualize the nuclei. Only Gr-1⁺ cells costained with the pDP4 antiserum. (E) pDP4 real-time RT-PCR of intestine RNA from PU.1^+/+ and PU.1^-/- mice at different embryonic (d.p.c.) and newborn (d.p.p.) stages. pDP4 values of 2 independent experiments are shown as percent 18S rRNA expression ± standard deviation.