pDP4 expression is induced during granulopoiesis in bone marrow but is down-regulated upon granulocytic mobilization. (A) Gr-1⁺/Mac-1⁺ granulocytic cells from bone marrow were further separated into mature Gr-1^high/Mac-1⁺ and immature Gr-1^low/Mac-1⁺ neutrophils using FACS sorting. (B) Quantitative real-time RT-PCR of pDP4 transcripts using RNA from FACS-sorted early (Gr-1^low/Mac-1⁺) or late (Gr-1high/Mac-1⁺) granulocytes from bone marrow or peritoneum of thioglycollate-induced mice. pDP4 values are shown as percent expression of 18S rRNA ± standard deviation. (C) Gr-1⁺ granulocytes were isolated from bone marrow and peripheral blood of either uninduced or G-CSF-injected mice using MACS sorting. Real-time RT-PCR values for pDP4 transcripts of 4 mice are shown as percent of 18S rRNA. (D) Western blot with 60 μg total cell extracts from mouse bone marrow and spleen together with MACS-sorted Gr-1⁺ cells from bone marrow or peritoneum of thioglycollate-treated mice. The membrane was incubated with an antiserum against pDP4 followed by an anti-actin antibody to demonstrate equal protein loading.