Expression of FKN and its cognate receptor by BL cells vs mononuclear phagocytes. (A) FKN RT-PCR products amplified from Mutu-BL cells (BL), Bcl-2 transfectants (BLBcl-2), and UV-induced Mutu-BL cells (BLUV). β-actin products are shown for comparison. (B) Expression of FKN on the surface of BL cells. BL2 cells were labeled by indirect immunofluorescence using anti-FKN mAb (clone 81513). Flow cytometric histograms of surface FKN (dark gray) compared with background immunostaining (light gray) are shown. (C) Immunoblots showing FKN expression in lysates of BL2 cell lines and BL tumors. SDS-PAGE (30 μg of protein per lane) was performed on lysates from BL2 cells (left panels) and 3 separate BL2 tumors grown in SCID mice (right panels). Proteins were transferred to Hybond P membrane and immunoblotted using anti-FKN antibody (clone 81513). Blots were stripped and reprobed for β-actin to check for loading variability. (D) Expression of FKN in tumor tissue in situ. Immunohistochemical staining was performed on archive paraffin sections of BL tumor tissue. Diaminobenzidine (DAB) chromogen product (brown) represents FKN staining (hematoxylin counterstain). Primary anti-FKN mAb (clone 81513, top panel); isotype control mAb (mouse IgG1, bottom panel). Arrow indicates example of starry-sky macrophage. Bar represents 52 μm. Stained tissue was analyzed using a Zeiss Axioskop 2 microscope (Zeiss, Welwyn Garden City, United Kingdom) at 20°C with objective lens Plan-NEOFLUAR 20× (numerical aperture 0.50) and histomount medium (Raymond A. Lamb, Eastbourne, United Kingdom), and images were taken using Jenoptik ProgRes C14 camera (Jenoptik, Jena, Germany) with Openlab 4.0.2 software (Improvision/PerkinElmer, Coventry, United Kingdom). (E) FKN receptor, CX3CR1 RT-PCR products amplified from HMDM (Mac), and the monocyte/macrophage cell lines THP-1 and MonoMac6 (MM6). β-actin products are shown for comparison. (F) Expression of FKN receptor, CX3CR1 on the surface of human monocytes, macrophages, and monocyte/macrophage cell lines THP-1 and MM6 but not BL cells (BL2 and Mutu-BL). Viable cells were labeled by indirect immunofluorescence using anti-CX3CR1 polyclonal antibody primary and PE-conjugated secondary. Flow cytometric histograms of surface FKN (dark gray) compared with background immunostaining (light gray) are shown.
