Expression of CD99L2 on endothelial cells. (A) Immunoblots of cell lysates of CHO cells stably transfected with the short E3—E4 splice variant (CD99L2_s; lanes 1-2 and 4-5), with the long E3A-E3-E4 splice variant of mouse CD99L2 (CD99L2_l; lane 6), or mock-transfected (lane 3), and of lysates of bEnd.5 endothelioma cells (lanes 7-8) were analyzed with preimmune serum IgG (co-IgG, lanes 1 and 7) or affinity purified antibodies against CD99L2 (anti-CD99L2, lanes 2-6, 8). To control the specificity anti-CD99L2 antibodies were preincubated either with recombinant CD99L2-Fc (lane 4) or with ESAM-Fc (lane 5). (B) Purified CD99L2-Fc was digested without enzymes or with neuraminidase (NA) or with neuraminidase and O-glycosidase (O-Gly), followed by immunoblotting with peroxidase-conjugated anti human IgG antibodies. (C) Detection of CD99L2 splice variants in bEnd.5 endothelioma cells and HUVEC by RT-PCR. (D) Immunofluorescence staining of bEnd.5 endothelioma cells with affinity purified antibodies against CD99L2 and negative control IgG from the respective preimmune serum (as indicated). First antibodies were detected with Cy3-conjugated secondary antibodies and visualized by fluorescence microscopy. Bar = 20 μm. (E) Immunoperoxidase staining of cryostat sections of mouse heart, kidney, and liver with affinity-purified antibodies against mouse CD99L2, mouse CD99, and control rabbit IgG from preimmune serum (as indicated). Tissue sections were incubated with first antibodies, followed by washing and incubation with secondary and tertiary reagent. Bar = 70 μm