Ephrin B2 but not EphB4 is expressed in KS biopsy tissue. (A) ISH with antisense probes for ephrin B2 and EphB4 with corresponding H&E-stained section to show tumor architecture. Dark blue color in the ISH indicates positive reaction for ephrin B2. No signal for EphB4 was detected in the KS biopsy. For contrast, ISH signal for EphB4 is strong in HNSCC tumor cells. Ephrin B2 was also detected in KS using EphB4-alkaline phosphatase (AP) fusion protein (iv). AS indicates antisense; S, sense. (B) Detection of ephrin B2 with EphB4/Fc fusion protein. Adjacent sections were stained with H&E (i) to show tumor architecture; the black rectangle indicates the area shown in the EphB4/Fc-treated section (ii) detected with FITC-labeled anti-human Fc antibody as described in “Patients, materials, and methods.” As a control, an adjacent section was treated with human Fc fragment (iii). Specific signal arising from EphB4/Fc binding to the section is seen only in areas of tumor cells. (C) Coexpression of ephrin B2 and the HHV-8 latency protein LANA1. (i-ii) Double-label confocal immunofluorescence microscopy with antibodies to ephrin B2 (red), LANA1 (green), or EphB4 (red) of frozen KS biopsy material directly demonstrates coexpression of LANA1 and ephrin B2 in KS biopsy. Coexpression is seen as yellow color. (iii) Double-label confocal image of biopsy with antibodies to PECAM-1 (green) in cells with nuclear propidium iodide stain (red), demonstrating the vascular nature of the tumor. (D) Western blot of protein extracts of KS biopsies, normal skin, and cell lines. Membranes were sequentially probed with ephrin B2, EphB4, and β-actin monoclonal antibodies. Specific bands were detected at 120 kDa for EphB4, 37 kDa for ephrin B2, and 40 kDa for β-actin. Oral squamous cell carcinoma cell lines SCC-15 an SCC-25 are included as positive controls for EphB4 probing. Membranes were probed with β-actin as a control for loading and transfer of protein.