Cloning and expression of CD99. (A) Deduced amino acid sequence of mouse CD99 (top sequence) and alignment with human CD99 (2 middle sequences) and human Xga protein (bottom sequence). Both splice variants of human CD99 (hCD99 type I and type II) are depicted and vary only at their C-terminus. Except for the cleavage site of the signal sequence of human CD99, which is based on sequencing of the mature CD99 protein,27 the putative signal sequences and the transmembrane regions (marked in boxes) were predicted by the SMART.EMBL program. ▪ indicates identical amino acids; ▪, homologous amino acids; and □, signal sequence (top panels) or transmembrane region (bottom panel). (Bi) Detection of mouse CD99 in Western blots. Cell lysates of CD99-transfected CHO cells (lanes 1-2, 4-5), mock-transfected CHO cells (lane 3), and mouse bEnd.5 endothelioma cells (lanes 6-7) were analyzed in immunoblots for reactivity with preimmune serum IgG (co-IgG: lanes 1, 6) or affinity-purified polyclonal antibodies against CD99 (anti-CD99: lanes 2-5, 7). To further control specificity, antibodies were mixed with either recombinant CD99-Fc (lane 4) or with ESAM-Fc (lane 5). (Bii) Lysates of CHO-CD99 cells or bEnd.5 cells were either mock incubated (–) or incubated with 5 mU sialidase from Arthrobacter ureafaciens (+) for 6 hours at 37°C prior to immunoblotting. Binding of first antibody was detected by incubating with peroxidase-conjugated secondary antibodies followed by visualization via chemiluminescence. (C) Cell-surface expression of CD99 on T cells (CD3ϵ⁺), B cells (CD19⁺), granulocytes (Gr-1high and 7/4⁺), and monocytes (Gr-1int and 7/4⁺) from peripheral blood mouse leukocytes (PBML) and of CD11c⁺ cells from mouse spleen was determined by 3-color FACS analysis. Negative staining was determined with control IgG from preimmune serum (gray line) and CD99 staining, with affinity-purified anti-CD99 antibodies (black lines).