JAM-B interaction with HSC is JAM-C-dependent. (A) Representative flow cytometry profiles showing the gating strategy used to define the hematopoietic progenitor subset. LSK cells are defined as Lin^NegSca^Posc-Kit^Pos cells and are further divided into three distinct populations on the basis of CD48 and CD150 expression as indicated. The most immature progenitors containing LT-HSC and MPP1 are defined as CD150^PosCD48^Neg LSK cells. (B) Histograms showing the binding of recombinant soluble JAM-B (black line) compared with sol-JAM-C (dashed line) or unstained cells (filled gray). Percentages of cells binding to sol-JAM-B are indicated. (C) Histograms showing JAM-C expression (black line) compared with control staining (dashed line). A rabbit polyclonal antibody was used and compared with the staining obtained with pre-immune serum. Mean fluorescence intensities (MFI) are indicated. (D) Left panel: Percentage of chimerism observed in the blood of lethally irradiated mice 4 weeks after engraftment with the indicated hematopoietic subsets. One representative experiment of 3 is shown (n = 6 mice/group; **P < .01). Right panel: Survival curves of mice engrafted with the indicated cells are shown. The survival curves of mice engrafted with total LSK and LSK sol-JAM-B^Pos are overlayed. (E) Representative flow cytometry profiles showing the levels of sol-JAM-B binding or JAM-C expression on LSK cells in the absence or presence of blocking pAb. JAM-B binding to LSK is prevented by rabbit-anti JAM-C antibody. This experiment was realized on at least 6 independent mice. (F) Representative flow cytometry profiles showing JAM-B binding and VLA-4 expression on JAM-C deficient LSK cells. JAM-B binding and VLA-4 expression on LSK cells isolated from Jam-C deficient (filled gray) or control animals (plain line) are compared with control (dashed line). This experiment was realized on at least 4 independent Jam-C deficient mice and compared with control littermate mice.