VCAM-1 expression in BM cells of several lineages and its absence from PB. (A) Dot blots of BM cells from control (f/f) and ablated (Δ/Δ) mice doubly labeled with anti-CD45, anti–c-kit, or lineage-affiliated markers (Gr1, Mac-1, B220, CD3, TER119) and with VCAM-1. Note the presence of VCAM-1 positivity in all lineages in normal BM cells and its virtual absence from VCAM-1–ablated animals. VCAM-1 expression is also seen at the RNA level (reverse transcriptase [RT]–PCR was performed on total RNA isolated from unsorted and sorted Gr1⁺/VCAM-1⁺ and Gr1⁺/VCAM-1^– bone marrow cells; see designated boxes). (B) Expression of VCAM-1 in BM or PB kit⁺ cells before and 5 days after G-CSF mobilization in normal (VCAM-1^f/f) mice. BM or PB cells were doubly labeled with anti–c-kit and anti–VCAM-1 and the expression levels of both are depicted in the dot blots. In BM, note the increase in kit⁺ cells, but the decreased proportion of VCAM-1⁺ cells after G-CSF–induced mobilization; in PB, note the absence of VCAM-1 positivity both before and after mobilization. Numbers in dot plots indicate the percentage of positive cells. (C) VCAM-1 RNA expression in mobilized and nonmobilized BM cells from a control animal. Total RNA was reverse transcribed and PCR for VCAM-1 was performed on serially diluted cDNA templates. Note the decrease in RNA expression for both 7-domain (VCAM-1 7D) and GPI-linked (VCAM-1/GPI) VCAM-1 isoforms in mobilized (+G/FL) versus nonmobilized (–G/FL) BM cells.