Antibody 440c recognizes Siglec-H. (A) Total splenocytes from WT (C57BL/6), DAP12^-/-, DAP10^-/-, or FcRγ^-/- mice (all on C57BL/6 background) were stained with phycoerythrin (PE)-labeled mAb anti-B220 and biotinylated mAbs 440c⁷  or mPDCA1,¹³  followed by streptavidin-APC and analyzed by 2-color flow cytometry. Percentages of 440⁺ and mPDCA1⁺ IPCs are indicated. IPCs appear to be less abundant in WT mice than in DAP12^-/- mice. It is possible that the lack of 440c Ag expression in the DAP12^-/- mice influences IPC homing to the spleen, IPC development, or IPC survival. (B) 293T cells expressing murine DAP12 (293T-DAP12) and 293T cells were transiently transfected with a Siglec-H-encoding cDNA, stained with mAb 440c, and analyzed by flow cytometry. FSC, forward scatter. (C) Alignment of V-type Ig domains of Siglec-H and mouse CD33. Identical residues are darkly shaded, similar residues are lightly shaded. Residues likely involved in sialic acid binding are boxed.¹⁶  (D) Alignment of transmembrane regions of mouse Siglec-H, mouse CD33, and chimpanzee Siglec-13. The conserved lysine residue that may allow for DAP12 association is indicated with an asterisk.