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. 2015 Aug 12;290(39):23997–24006. doi: 10.1074/jbc.M115.653378

FIGURE 2.

FIGURE 2.

IL-36 treatment alters the localization of the IL-36R. A, flow cytometry analysis of the IL-1Rrp2 present on the cell surface and within cells. NCI cells were treated or mock-treated with IL-36γ for 30 min. Cell surface-localized IL-1Rrp2 was determined in fixed NCI cells that were not permeabilized. Intracellular IL-1Rrp2 was determined by the difference in the total signal for IL-1Rrp2 subtracted from the signal present on the cell surface. All samples were tested in triplicates, with the signal from 10,000 cells enumerated. B and C, effects of endocytosis inhibitors on IL-36R signaling. Shown is IL-6 production in NCI cells treated with IL-36γ in the presence of increasing concentrations of the clathrin-independent endocytosis inhibitor MβCD or the clathrin-dependent endocytosis inhibitor chloropromazine. IL-6 production by NCI cells was used as a control. D, IL-1Rrp2 colocalized with clathrin after the addition of IL-36γ. NCI cells were fixed and stained with antibodies to detect IL-1Rrp2 (red), clathrin (green), or DNA (blue). Colocalization of IL-36R and clathrin is pseudocolored yellow. The percent colocalization is shown in the top left corners of the micrographs, which are representative for the analyzed conditions. A minimum of 20 cells were quantified in each sample. E, quantified colocalization of IL-1Rrp2 with Alexa Fluor 594-labeled human transferrin receptor or cholera toxin B (CTxB). The hTFN receptor trafficked into cells using clathrin-mediated endocytosis, whereas cholera toxin B traffics into cells using a clathrin-independent endocytosis marker. Percent colocalization was quantified from more than 20 independent cells from three independently prepared samples. F, IL-36R signaling does not require endosome acidification. IL-36γ induced IL-6 production in NCI cells in the presence of increasing concentrations of the endosomal acidification inhibitor bafilomycin A1. All data are the mean of three independent experiments where the IL-6 levels secreted by the cells into the medium were normalized for the mock-treated NCI cells.