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. 2014 Nov 5;290(2):861–871. doi: 10.1074/jbc.M114.586560

FIGURE 2.

FIGURE 2.

IL-17RD is critical for NF-κB activation in RTECs. A, overexpression of IL-17RD enhances the NF-κB transcriptional activity in HK-2 cells. HK-2 cells transfected with NF-κB luciferase reporter, pRL-TK, and Myc-IL-17RD or Myc-TNFR2 were stimulated with TNF-α for 6 h. Results presented are the average from a triplicate experiment. **, p < 0.01. B and C, depletion of IL-17RD suppresses NF-κB activation in HK-2 cells. After depletion of IL-17RD using adenovirus-driven shRNA against IL-17RD (named Ad-hIL-17RDi-1 and Ad-hIL-17RDi-2), the IL-17RD mRNA was examined by an RT-PCR. Adenovirus-driven shRNA against GFP (named Ad-GFPi) was used as a control. GAPDH was used as a control. The ratio represents the relative density of IL-17RD over GAPDH (B). HK-2 cells were transfected with NF-κB luciferase reporter, pRL-TK, and infected with indicated adenovirus. *, p < 0.05; ***, p < 0.001 (C). D, depletion of hIL-17RD suppressed the phosphorylation levels of IκB-α in HK-2 cells. Bottom, densitometry of the bands, showing the p-IκB-α level, normalized to β-actin, compared with Ad-GFPi control. *, p < 0.05. Data are representative of at least three experiments. E and F, depletion of hIL-17RD inhibits the TNF-α-induced p65 nuclear translocalization. HK-2 cells affected with Ad-hIL-17RDi-1 and Ad-GFPi were stimulated with TNF-α for 5 min. The cytoplasmic (Cyt) and nuclear (Nuc) fractions of HK-2 cells were extracted and blotted with an anti-p65 antibody (E). Quantification of the ratio of nuclear/cytoplasmic p65, normalized by tubulin and c-Jun respectively, are shown (F). G, IL-17RDY330F abrogates the IL-17RD inhibition of TNF-α-induced NF-κB in HEK293T cells. Cells were treated as in A. H, depletion of IL-17RD promotes TNF-α-induced NF-κB transcriptional activity in HEK293T cells. I, a Western blot assay for the protein levels of IL-17RD and TNFR2 or TNFR1 in HEK293T and HK-2 cells. J, TNFR2 suppresses the association of IL-17RD and p65 in HEK293T cells. The protein complex was immunoprecipitated (IP) by an anti-FLAG antibody and resolved by an anti-Myc or anti-FLAG antibody. IB, immunoblot. K, IL-17RD fails to interact with NF-κB (p65) in HK-2 cells. An endogenous IP experiment was performed for HK-2 cells. L, immunohistochemical staining for TNFR2 and TNFR1 in sections from kidneys of IgA nephropathy and its control (bars, 500 μm).