Figure 5. Binding of soluble chemokines to corresponding tm-chemokines on cell surfaces.
(A) Fluorescent soluble chemokines, either labeled directly (cyanine3, Cy3) or indirectly via biotin-label to fluorescent (strept)avidin (Alexa-Fluor 488 or fluorescein-isothiocyanate, FITC), bind to tumor cells expressing tm-chemokines (transfected LOX melanoma, or glioma cells, not shown), but not to non-transfected LOX cells as shown by light microscopy. Due to its lipophilic character, the dye Cy3 yields a whiff of background. However, this was also observed with the negative control Cy3-labeled lactalbumin. Chemokine concentrations 2 nM, bars indicate 20 µm. (B) FACS analysis confirmed binding signals yielded by fluorescence microscopy. LOX cells transfected with tm-CXCL16 were detectable by their binding of biotinylated CXCL16 (2 nM)/Avidin-FITC, whereas mock transfected cells (LOX-pcDNA) were not, and LOX cells transfected with tm-CX3CL1 were labeled by biotin-CX3CL1 (2 nM)/Avidin-FITC, in comparison to mock transfected cells. (C) A close association between s- and tm-chemokines is visible by immuno-electron microscopy of tm-chemokine-expressing/-transfected cells which were immunolabeled with anti-CXCL16 or CX3CL1 and 15 nm-gold-labeled secondary antibodies and subsequently incubated with 5 nm-gold-labeled (directly or via biotin/streptavidin complex) soluble chemokines (2 nM) before embedding. (D) Chemical cross-linking by paraformaldehyde (PFA) shifts tm-chemokine bands to higher molecular weights. Isolated membranes of tm-chemokine overexpressing LOX cells were incubated over night with 2 nM s-chemokines, cross-linked with 1% PFA and subjected to SDS-PAGE separation and subsequent Western blotting using antibodies against the respective chemokine domains of CXCL16 and CX3CL1. All experiments were repeated in 2 independent biological replicates, and representative photographs and immunoblots are shown.