PMC:4419340 / 14192-18649 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4419340","sourcedb":"PMC","sourceid":"4419340","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4419340","text":"Impaired production of IL17RC mRNA and protein\nWe used RT-PCR or TaqMan assays to determine the levels of full-length IL17RC mRNA, which we found to be lower in fibroblasts from the patients than in cells from healthy controls (Fig. 4, A and B). However, no band of the correct size corresponding to IL-17RC could be detected by Western blot in control fibroblasts without transfection. Therefore, the patients’ fibroblasts were then transfected with various expression vectors: an empty vector or vectors encoding the WT or any of the three mutant alleles (Q138*, R376*, or R378*). In this overexpression system, the IL17RC mRNAs generated by transcription from the WT allele or the three mutant alleles were detected equally well by qPCR (Fig. 4 C). A product of ∼85 kD, corresponding to isoform 1, was detected in cells transfected with the WT IL17RC allele, whereas no product of this molecular mass was detected in fibroblasts transfected with any of the three mutant IL17RC alleles (Fig. 4 D). No product with a lower molecular mass was detected in cells transfected with the Q138* IL-17RC–encoding construct, with an anti–IL-17RC antibody directed against the epitope covering amino acids 113–258. A product with a lower molecular mass (∼40 kD), corresponding to a C-terminally truncated protein, was detected in cells transfected with the constructs encoding R376* or R378* IL-17RC. A product of ∼80 kD was detected in cells transfected with the WT allele and a product of ∼35 kD was detected in cells transfected with the R376* or R378* allele. These products were probably the result of posttranscriptional modifications. In addition, HEK-293T cells transfected with various constructs encoding IL-17RC WT, Q138*, R376*, or R378* displayed a strong membrane expression, as detected by total internal reflection fluorescence (TIRF) imaging, only when transfected with the WT allele but none of the mutants alleles (Fig. 5 A). The absence of IL-17RC had no impact on the expression of IL-17RA, which was found to be expressed to similar levels on the fibroblasts of patients and controls (Fig. 5 B).\nFigure 4. Expression of IL-17RC in fibroblasts from controls and patients. (A and B) Amounts of IL17RC cDNA generated from SV40-immortalized fibroblasts from two controls and two patients (P1 and P2), as determined by full-length RT-PCR (A) and TaqMan assays (B). (C) Amounts of IL17RC cDNA obtained from the SV40 fibroblasts of P1 and P2 either left untransfected (NT) or transfected with pUNO1, either empty (MCS) or encoding the WT, Q138*, R376*, or R378* IL-17RC. Results are also shown for the SV40 fibroblasts of two controls tested in parallel (C1 and C2). Means ± SD (error bars) of three independent experiments, as detected by quantitative PCR, are shown. β-ACTIN and GUS were used as endogenous controls. The experiments were repeated at least three times. (D) IL-17RC expression in P1’s and P2’s SV40 fibroblasts transfected with WT or mutant IL17RC alleles, as assessed by Western blotting. IL-17RC protein levels in SV40 fibroblasts from P1 and P2 transfected with the empty pUNO1mcs plasmid (mock) or the pUNO1 plasmid, encoding the WT or one of the three mutant (Q138*, R376*, or R378*) IL-17RC proteins, as determined by Western blotting with an anti–IL-17RC antibody (directed against amino acids 113–258). The anti-GAPDH antibody was used as a control for protein loading. These experiments were repeated at least three times.\nFigure 5. Expression of WT, mutant IL-17RC, and IL-17RA at the cell surface. (A) IL-17RC expression in HEK-293T cells transfected with V5 tag plasmid encoding IL-17RC WT or mutant alleles, as assessed by TIRF imaging. HEK-293T cells were transfect with V5 tag–IL17RC-pcDNA 3.1 encoding WT or mutant (Q138*, R376*, or R378*) IL-17RC. The “Epi” was assessed by epifluorescence illumination, and the “TIRF” was detected by TIRF microscopy. DAPI binds double-stranded DNA, and phalloidin binds F-actin. The “pseudocolor” scales were used to indicate the intensity staining in TIRF. For each setting condition, 20 cells have been analyzed from cumulating three independent experiments. Bars, 5 µm. (B) IL-17RA expression on SV40-immortalized fibroblasts from a control, P1 (Q138*/Q138*), P2 (R376*/R376*), and an IL-17RA–deficient patient (Q284*/Q284*), as assessed by flow cytometry. Isotype control, gray dotted lines; IL-17RA antibody, black lines. The experiments were repeated at least three times.\n\nI","divisions":[{"label":"Title","span":{"begin":0,"end":46}},{"label":"Figure caption","span":{"begin":2108,"end":3456}},{"label":"Figure caption","span":{"begin":3455,"end":4456}}],"tracks":[]}