FIGURE 4.

Tollip participates in IL-36R signaling and endocytosis. A, the effect of Tollip knockdown on IL-6 production by the IL-36R and the IL-1R in NCI and NCI-KO cells. siRNA knockdown used control siRNAs (C) or siRNAs specific to Tollip (T). IL-6 produced in response to LPS served as a control for the responsiveness of the cells. *, p < 0.05. B, IL-1Rrp2 colocalizes with Tollip after IL-36γ addition. The cells were fixed and processed for immunofluorescence confocal microscopy 30 min after the addition of IL-36γ or IL-36RA. C, quantification of the colocalization of Tollip and IL-1Rrp2 in NCI cells after addition of IL-36γ and IL-36RA at different time points. The data were derived from immunoconfocal microscopy of cells stained with antibodies to detect IL-1Rrp2 or Tollip. Student's t test was used to calculate the degree of colocalization in cells treated with different ligands. *, p < 0.05. D, IL-1Rrp2 coimmunoprecipitates with Tollip. Shown are images from Western blots probed with antibodies to detect IL-1Rrp2, the loading control β-actin, and the amount of IL-1Rrp2. The cells were also treated with siRNAs to either controls or Tollip. Immunoprecipitations used a monoclonal antibody specific to Tollip.