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. 2017 Nov 25;10(1):56–69. doi: 10.1159/000481210

Fig. 5.

Fig. 5

HEK293T cells reconstituted for IL-36R signalingrespond to RNF125. a RNF125 overexpression in HEK293T cells increased the polyubiquitination of activated IL-1Rrp2. Cells were transfected with plasmids expressing IL-1Rrp2 (1 μg) and RNF125 (1.0 μg) for 36 h, and the degree of IL-1Rrp2 polyubiquitination was determined by immunoprecipitation of IL-1Rrp2 and Western blot to detect ubiquitin. Control IgG was used to determine the degree of nonspecific binding and no polyubiquitinated IL-Rrp2 was observed. All Western blot data shown are representative of 3 independent experiments. b Effect of RNF125 knockdown on IL-36R signaling in HEK293T cells. Cells were transfected with 50 nM of siRNA specific to RNF125 or control siRNAs. Cells were incubated for 48 h following siRNA transfection. All data represent means of 3 independent experiments. p values were calculated using the Student t test. Western blots (above) depict the accumulation of RNF125 and the β-actin loading controls in HEK293T cells knocked down with siRNAs specific to RNF125 or with control siRNAs. c Overexpression of RNF125 or RNF125 with a C-terminal Myc-tag increased IL-36R signaling in HEK293T cells. IL-36R signaling was assessed with the activity of luciferase driven by an NF-κβ promoter element and normalized to the constitutive Renella luciferase expressed from the same cells. All cells were transfected with 1 ng of plasmid expressing IL-1Rrp2 along with an increasing concentration of RNF125-expressing plasmid and incubated for 3 h. The amount of plasmid transfected into cells was normalized using the empty vector plasmids. The cells were treated with IL-36γ (1 ng/mL) for 12 h prior to assessment of the luciferase activity. d RNF125 overexpression decreases IL-1Rrp2 accumulation in HEK293T cells in a concentration-dependent manner. The abundance of RIG-I was assessed to demonstrate that the overexpression had the expected effect, as RNF125 is known to decrease RIG-I abundance. Cells were incubated for 48 h following transfection and the plasmid concentration was normalized by individual empty vector plasmids. e RNF125 forms a coimmunoprecipitable complex with IL-1Rrp2. Coimmunoprecipitation used mAb to the Myc-epitope and control IgG. HC denotes the heavy chain of the IgG used in the immunoprecipitation assay.