Plasma membrane binding of IFI16 to HUVEC.

<p>(A) Cells were left untreated (a, negative control) or incubated with increasing concentrations of FITC-labeled recombinant IFI16 (b, 10 nM rIFI16-FITC; c, 20 nM rIFI16-FITC; d, 30 nM rIFI16-FITC). Binding was detected by confocal microscopy using an excitation wavelength of 490 nm for FITC in one channel and trans-illuminated light in the other. Representative images of three independent experiments are shown. (B) Endogenous IFI16 released by irradiated HeLa cells binds neighboring HUVEC. UV-B irradiated HeLa cells were co-cultured with HUVEC and after 24 h, 36 h and 48 h, dead cell debris were removed and immunofluorescence was performed on the remaining live HUVEC using a home-made anti-IFI16 polyclonal as primary antibody and Alexa-488- anti-rabbit as secondary antibody. The cells were then fixed, permeabilized, nuclear-stained using propidium iodide and analyzed by confocal microscopy. Fibroblasts were employed as negative control. Representative images of three independent experiments are shown.