Vascular and lymphatic endothelial cells express multiple isoforms of CLEVER-1. (A) The indicated cell (2 different HDMEC populations containing both vascular and lymphatic endothelial cells [mixed], pure vascular and pure lymphatic fractions of HDMECs, and HUVECs) and tissue lysates (tonsil) were analyzed by immunoblotting for CLEVER-1 protein. Molecular weight (Mw) markers in kilodaltons are indicated on the left. (B) Different isoforms of CLEVER-1 contain posttranslational modifications in endothelial cells. Lysates from lymphatic (pure lymphatic fraction of HDMECs) and vascular (HUVECs) endothelial cells were left untreated (-), digested with sialidase (Sial), or digested with sialidase followed by O-glycanase (Sial + O-glyc) and analyzed by immunoblotting. (C) The presence of the variably spliced exon 27 in the samples of lymphatic and vascular HDMECs, and HUVECs was analyzed by RT-PCR. The upper band represents the exon 27-containing transcript of the CLEVER-1 gene, whereas the lower band indicates the presence of CLEVER-1 mRNA isoform in which the exon 27 has been spliced out. Representative results of 2 independent experiments are shown.