sandwich enzyme linked immunosorbent assay elisa kits  (R&D Systems)

Journal: Life Science Alliance

Article Title: Dectin-1 epigenetic reprogramming rescues senescent-like Treg function in allergic asthma

doi: 10.26508/lsa.202503552

Figure Lengend Snippet: FACS-sorted Tregs from patients with asthma and healthy controls (n = 25 per group for all panels) were evaluated for suppressive capacity, intracellular cytokine expression, and secreted cytokine production. (A, B) Suppressive capacity. Representative flow cytometry plots of responder T-cell proliferation and quantitative analysis of Treg-mediated suppression. Tregs from asthma patients exhibited reduced suppressive efficiency compared with controls. (C) Intracellular cytokine expression. Frequencies of Tregs expressing anti-inflammatory cytokines (IL-10, TGF-β) and pro-inflammatory cytokines (IFN-γ, IL-17). Asthma-derived Tregs showed decreased IL-10 + and TGF-β + populations and increased IFN-γ + and IL-17 + populations relative to controls. (D) Secreted cytokine production. Concentrations of IL-10 and active TGF-β1 in 72-h culture supernatants from highly purified Tregs. Asthma Tregs secreted lower levels of both cytokines compared with healthy controls. Data are presented as the mean ± SD, with each dot representing an individual sample. Statistical significance was determined using Welch’s unpaired t test or one-way ANOVA followed by Tukey’s post hoc test, as appropriate. For panels (B, C), P -values were Bonferroni-corrected for multiple comparisons. Significant differences are indicated by asterisks (**** P < 0.001).

Article Snippet: Human IL-10, TGF-β1, IFN-γ, and IL-17 ELISA kits (R&D Systems) were used for the detection of cytokines.

Techniques: Expressing, Flow Cytometry, Derivative Assay, Purification

Journal: Life Science Alliance

Article Title: Dectin-1 epigenetic reprogramming rescues senescent-like Treg function in allergic asthma

doi: 10.26508/lsa.202503552

Figure Lengend Snippet: (A, B) Purity and batch consistency analysis of KQS-1. HPLC analysis of KQS-1 production batches. (A) Quantitative analysis of purity across three independent production batches (n = 5 replicates per batch). Individual data points (black dots) represent single measurements, and the vertical distribution shows batch uniformity. The red dashed line denotes the minimum purity requirement of 95%. Statistical analysis by one-way ANOVA indicates no significant variation between batches (ns, P > 0.05), confirming high-level production consistency. (B) Representative HPLC chromatogram overlay of three batches showing consistent peak profiles and identical retention times, confirming the chemical stability and absence of batch-to-batch variation in the manufacturing process. (C, D, E) Impact of PBS vehicle on the function and viability of asthma patient–derived Tregs. Assessment of PBS treatment on FACS-sorted CD4 + CD25 + CD127 − Tregs isolated from asthma patients (n = 25 patients per group). (C) Treg suppressive efficiency (%) measured by the inhibition of responder T-cell proliferation. No significant difference was observed between the untreated control and the PBS vehicle group. (D) Frequencies (%) of intracellular anti-inflammatory (IL-10, TGF-β) and pro-inflammatory (IFN-γ, IL-17) cytokines determined by flow cytometry after 48 h. Comparisons were adjusted using the Bonferroni correction; no significant alterations were detected across all markers. (E) Treg survival rate (%) after 48 h of culture in the presence or absence of PBS. In all panels, box-and-whisker plots represent the median, interquartile range (IQR), and range, whereas individual black dots denote data from independent patient samples. Statistical significance was evaluated using Welch’s unpaired t test. ns indicates P > 0.05, confirming that PBS serves as a neutral vehicle for KQS-1.

Article Snippet: Human IL-10, TGF-β1, IFN-γ, and IL-17 ELISA kits (R&D Systems) were used for the detection of cytokines.

Techniques: Derivative Assay, Isolation, Inhibition, Control, Flow Cytometry, Whisker Assay

Journal: Life Science Alliance

Article Title: Dectin-1 epigenetic reprogramming rescues senescent-like Treg function in allergic asthma

doi: 10.26508/lsa.202503552

Figure Lengend Snippet: (A) Suppression assays demonstrating that KQS-1–treated Tregs exhibit enhanced inhibition of responder T-cell proliferation compared with vehicle-treated controls. (B, C) Cytokine profiling after 48-h KQS-1 exposure. ELISA analysis of culture supernatants shows a shift toward an anti-inflammatory phenotype, characterized by increased IL-10 and TGF-β1 production and reduced IFN-γ and IL-17 levels. (D) Treg survival analysis in patient-derived cells. Representative and quantitative assessment of Annexin V + /PI − apoptotic Tregs after 48-h treatment with KQS-1 or vehicle control. Paired samples are connected by lines. Data are presented as the mean ± SD. (A, B, C, D) Sample sizes: n = 8 (A, B, C) and n = 12 (D). Statistical significance was determined using unpaired or paired two-tailed t tests, as appropriate, with Bonferroni correction for multiple comparisons where applicable. ** P < 0.01, *** P < 0.001.

Article Snippet: Human IL-10, TGF-β1, IFN-γ, and IL-17 ELISA kits (R&D Systems) were used for the detection of cytokines.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Derivative Assay, Control, Two Tailed Test

Journal: PLOS One

Article Title: Clinical proof of concept for small molecule mediated inhibition of IL-17 in psoriasis

doi: 10.1371/journal.pone.0341049

Figure Lengend Snippet: (A) Inhibition of IL-17 bioactivity as secreted by human TH17 cells and assayed by a reporter cell assay. Dotted lines represent positive and negative assay controls. 1 representative of 7 experiments with different donors is shown. The error bars represent the SEM. (B, C) Evaluation of DC-806 in rat CIA. Rats were randomized on Day 11 and dosed as indicated. Daily measurements of ankle thickness (B) and terminal measurement of footpad weights (C) were used as efficacy readouts. (D) Preclinical evaluation of DC-806 serum levels. PK sampling for exposure determination was performed on Days 11, 16, at 1 and 12 hours post dose. The dotted lines represent the uncorrected IC 50 for rat IL-17AA and rat IL-17AF as listed in . The dashed line represents the LLQ of the quantification assay. All error bars represent the SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Abbreviations : anti-F, anti-IL-17F; BID, twice daily; CIA, collagen-induced arthritis; Dex, dexamethasone; IC 50 , half-maximal inhibitory concentration; IL-17, interleukin-17; LLQ, lower limit of quantification; PK, pharmacokinetic; QD, once daily; SEM, standard error of mean; TH17, T-helper 17.

Article Snippet: Serum IL-17A level was measured using Quantikine ® High Sensitivity (HS) Human IL-17 Immunoassay kit #HS170 (R&D Systems), which mostly detects IL-17AA with approximately 3% cross-reactivity to IL-17AF [ ].

Techniques: Inhibition, Sampling, Concentration Assay

Journal: PLOS One

Article Title: Clinical proof of concept for small molecule mediated inhibition of IL-17 in psoriasis

doi: 10.1371/journal.pone.0341049

Figure Lengend Snippet: Graph detailing the group mean ± SD DC-806 plasma concentration by 200 mg BID (green circle) and 800 mg BID (blue diamond) groups on a semilogarithmic scale through Study Day 29. PK in psoriasis patients was comparable to healthy volunteer PK, with consistent trough levels over 28 days after achieving steady state around Day 3. The dotted line represents IC 50 for recombinant human IL-17AA, as obtained using HEK-blue IL-17 cell-based assay . PK Concentration Analysis Set. Abbreviations : BID, twice daily; HEK: human embryonic kidney; IC 50 , half-maximal inhibitory concentration; IL-17, interleukin-17; PK, pharmacokinetic; SD, standard deviation.

Article Snippet: Serum IL-17A level was measured using Quantikine ® High Sensitivity (HS) Human IL-17 Immunoassay kit #HS170 (R&D Systems), which mostly detects IL-17AA with approximately 3% cross-reactivity to IL-17AF [ ].

Techniques: Clinical Proteomics, Concentration Assay, Recombinant, Cell Based Assay, Standard Deviation

Journal: PLOS One

Article Title: Clinical proof of concept for small molecule mediated inhibition of IL-17 in psoriasis

doi: 10.1371/journal.pone.0341049

Figure Lengend Snippet: (A–C) DC-806 demonstrated biological effects as evidenced by dose-dependent responses of biomarkers of interest compared to placebo. Blue diamonds represent the 800 mg BID group; Green circles represent the 200 mg BID group; Black triangles represent the placebo group. (A) Change in mean ± SEM serum IL-17AA levels from time of first dose through 28 days post first dose. The dotted line represents low limit of quantification of the assay. (B) Change in mean ± SEM serum BD-2 levels from time of first dose through 28 days thereafter. *Except on Day 28 when n = 10; **Except on Day 28 when n = 9. (C) Change in mean ± SEM plasma IL-19 levels from time of first dose through 28 days post first dose. The dotted line represents normalization value of 21 pg/mL. PD Biomarker Analysis Set. Abbreviations: BD-2, beta defensin-2; BID, twice daily; BL, baseline; IL-17, interleukin-17; PASI, psoriasis area and severity index; PD, pharmacodynamic; SEM, standard error of mean.

Article Snippet: Serum IL-17A level was measured using Quantikine ® High Sensitivity (HS) Human IL-17 Immunoassay kit #HS170 (R&D Systems), which mostly detects IL-17AA with approximately 3% cross-reactivity to IL-17AF [ ].

Techniques: Clinical Proteomics, Biomarker Discovery

Journal: Research

Article Title: ZDHHC14-Mediated TEAD4 Palmitoylation Drives Th17 Cell Recruitment in Renal Immunopathology

doi: 10.34133/research.0954

Figure Lengend Snippet: Chemokine CCL20 recruits Th17 cells and is regulated by the transcription factor TEAD4. (A) ELISA results demonstrate elevated CCL20 expression levels in inflamed renal tubular epithelial cells (TECs) compared to normal TECs ( n = 3). (B and C) Transwell migration assays revealed a marked increase in the number of Th17 cells migrating toward inflamed renal TECs, as visualized by calcein staining (green fluorescence; n = 3; scale bar, 50 μm). The graph on the right quantifies the average number of migrating Th17 cells per field. (D) JASPAR analysis predicted potential TEAD4 binding sites within the promoter region of CCL20, suggesting a regulatory interaction. (E) Schematic representation of the construction of TEAD4 overexpression vectors and CCL20 promoter luciferase reporter gene vectors. (F) Luciferase reporter assays in 293T cells showed that TEAD4 overexpression substantially enhances CCL20 promoter activity, supporting its role as a transcriptional activator ( n = 3). (G) DNA pull-down assays subsequently confirmed a direct physical interaction between the CCL20 promoter and TEAD4 protein. (H) This binding was further validated by chromatin immunoprecipitation (ChIP) assays, which demonstrated the specific recruitment of TEAD4 to the endogenous CCL20 promoter region ( n = 3). (I) Proposed mechanism illustrating how TEAD4 promotes Th17 cell recruitment by up-regulating CCL20 transcription. Comparisons between 2 groups are performed using Student’s t test, whereas differences among 3 or more groups are assessed by one-way ANOVA. All results are expressed as mean ± SD. Statistical significance is denoted as follows: ns, not significant; ** P < 0.01; **** P < 0.0001. The schematic diagrams in (E) and (I) are created using BioRender.com under a valid license.

Article Snippet: Human serum IL-17 and CCL20 levels were measured using ELISA kits (R&D Systems, USA), with absorbance recorded at 450 nm and background corrected at 540 nm.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Migration, Staining, Fluorescence, Binding Assay, Over Expression, Luciferase, Activity Assay, Chromatin Immunoprecipitation

Journal: Research

Article Title: ZDHHC14-Mediated TEAD4 Palmitoylation Drives Th17 Cell Recruitment in Renal Immunopathology

doi: 10.34133/research.0954

Figure Lengend Snippet: Palmitoylation of TEAD4 at cysteine 367 (C367) is critical for its transcriptional regulation of CCL20. (A) Probable palmitoylation sites of TEAD4 in humans and mice. (B and C) The Click-iT assay revealed that the C367S (human) mutation in TEAD4 completely abrogated its palmitoylation ( n = 3). (D) The dual-luciferase reporter assays revealed that the C367S (human) mutation in TEAD4 reduced its transcriptional regulation of CCL20 ( n = 3). (E and F) ChIP followed by RT-qPCR analysis revealed that the C360S (mouse) mutation substantially diminished the binding occupancy of TEAD4 at the endogenous CCL20 promoter region ( n = 3). (G) Consistent with this transcriptional defect, ELISA confirmed that the C360S (mouse) mutation results in substantially decreased CCL20 protein expression ( n = 3). (H) As a direct functional consequence, Transwell migration assays demonstrated that expression of the TEAD4-C360S mutant in renal TECs markedly impaired the recruitment of Th17 cells (calcein AM staining, green fluorescence; n = 3; scale bar, 50 μm). Differences among 3 or more groups are assessed by one-way ANOVA. All results are expressed as mean ± SD. Statistical significance is denoted as follows: ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Human serum IL-17 and CCL20 levels were measured using ELISA kits (R&D Systems, USA), with absorbance recorded at 450 nm and background corrected at 540 nm.

Techniques: Mutagenesis, Luciferase, Quantitative RT-PCR, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Functional Assay, Migration, Staining, Fluorescence

Journal: Research

Article Title: ZDHHC14-Mediated TEAD4 Palmitoylation Drives Th17 Cell Recruitment in Renal Immunopathology

doi: 10.34133/research.0954

Figure Lengend Snippet: Fatty acid synthase (FASN) regulates palmitic acid synthesis, providing substrate for TEAD4 palmitoylation. (A and B) Single-cell sequencing analysis demonstrated differential FASN expression levels in renal tissues, including proximal tubular cells (PT cells), from IgAN patients. (C and D) Immunofluorescence (IF) staining showed the expression of FASN in kidneys of BSA nephritis mice (FASN, Cy3-labeled, red fluorescence; LTL, FITC-labeled, green fluorescence; n = 6; scale bar, 20 μm). Semiquantitative analysis of FASN-positive areas is presented on the right. (E and F) Click-iT assays revealed that TEAD4 palmitoylation was diminished in inflamed renal TECs following FASN knockdown, whereas, conversely, it was enhanced by FASN overexpression ( n = 3). (G) Reflecting this change in modification, luciferase reporter assays showed that FASN knockdown impaired TEAD4-mediated transcriptional activation of the CCL20 promoter, while FASN overexpression potentiated it ( n = 3). (H) Consistent with the transcriptional data, ELISA confirmed that CCL20 protein secretion was decreased following FASN knockdown and increased upon FASN overexpression ( n = 3). (I and J) Transwell migration assays indicated that FASN knockdown markedly reduced the number of Th17 cells migrating toward inflamed renal TECs. Transwell migration assays indicated that FASN knockdown substantially reduced the number of Th17 cells migrating toward inflamed renal TECs; notably, this reduction was rescued by the addition of recombinant CCL20. Conversely, FASN overexpression augmented Th17 migration, an effect that was reversed by a CCL20 neutralizing antibody (calcein AM staining, green fluorescence; n = 3; scale bar, 50 μm). The graph on the right quantifies the average number of migrating cells per field. Comparisons between 2 groups are performed using Student’s t test, whereas differences among 3 or more groups are assessed by one-way ANOVA. All results are expressed as mean ± SD. Statistical significance is denoted as follows: ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Human serum IL-17 and CCL20 levels were measured using ELISA kits (R&D Systems, USA), with absorbance recorded at 450 nm and background corrected at 540 nm.

Techniques: Sequencing, Expressing, Immunofluorescence, Staining, Labeling, Fluorescence, Knockdown, Over Expression, Modification, Luciferase, Activation Assay, Enzyme-linked Immunosorbent Assay, Migration, Recombinant

Journal: Research

Article Title: ZDHHC14-Mediated TEAD4 Palmitoylation Drives Th17 Cell Recruitment in Renal Immunopathology

doi: 10.34133/research.0954

Figure Lengend Snippet: ZDHHC14 is the key palmitoyltransferase (PAT) promoting TEAD4 palmitoylation and is highly expressed in the kidneys of IgAN patients and BSA nephritis mice. (A) RNA-sequencing analysis revealed a marked up-regulation of ZDHHC14 expression in the renal tissues of BSA nephritis mice. (B) Molecular docking predicted potential binding sites and binding affinity between ZDHHC14 and TEAD4, suggesting a direct interaction. (C and D) Co-immunoprecipitation (Co-IP) assays confirmed the interaction between TEAD4 and ZDHHC14 in renal TECs following inflammatory injury. (E) IHC staining showed substantially higher ZDHHC14 expression in the renal tissues of IgAN patients compared to adjacent nontumor tissue ( n = 3). Scale bars, 200 μm (upper panel) and 20 μm (lower panel). (F and G) ABE assays demonstrated that ZDHHC14 knockdown [via small interfering RNA (siRNA)] reduced, while ZDHHC14 overexpression increased, TEAD4 palmitoylation levels in inflamed renal TECs ( n = 3). (H) Dual-luciferase reporter assays revealed that ZDHHC14 knockdown decreased, while ZDHHC14 overexpression enhanced, TEAD4-mediated transcriptional activity on the CCL20 promoter in inflamed renal TECs ( n = 3). (I) ELISA results indicated that ZDHHC14 knockdown reduced, while ZDHHC14 overexpression increased, CCL20 levels in inflamed renal TECs ( n = 3). Differences among 3 or more groups are assessed by one-way ANOVA. All results are expressed as mean ± SD. Statistical significance is denoted as follows: ns, not significant; * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: Human serum IL-17 and CCL20 levels were measured using ELISA kits (R&D Systems, USA), with absorbance recorded at 450 nm and background corrected at 540 nm.

Techniques: RNA Sequencing, Expressing, Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunohistochemistry, Knockdown, Small Interfering RNA, Over Expression, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: ZDHHC14-Mediated TEAD4 Palmitoylation Drives Th17 Cell Recruitment in Renal Immunopathology

doi: 10.34133/research.0954

Figure Lengend Snippet: The PAT inhibitor 2-BP reduces Th17 cell infiltration and fibrosis progression in BSA nephritis mice. (A and B) To confirm their direct effects on TEAD4 modification, Click-iT assays showed that 2-BP treatment decreased TEAD4 palmitoylation, whereas, conversely, the depalmitoylase inhibitor ML349 enhanced its levels ( n = 3). (C) Dual-luciferase reporter assays demonstrated that treatment with the PAT inhibitor 2-BP markedly decreased TEAD4-mediated transcriptional activity on the CCL20 promoter in inflamed renal TECs, whereas treatment with the depalmitoylase inhibitor ML349 had no substantial effect ( n = 3). (D) ELISA results revealed that 2-BP treatment markedly reduced CCL20 expression levels in inflamed renal TECs, while ML349 treatment showed no substantial impact ( n = 3). (E) A schematic illustration delineated the intervention protocol utilizing 2-BP in BSA nephritis mice. Following a 12-week disease induction period, mice assigned to the treatment cohort received intraperitoneal administration of 2-BP at a dosage of 40 mg/kg, delivered once per week for an additional 12 weeks. In parallel, control animals were given an equal volume of vehicle solution according to the same schedule, thereby ensuring consistency between experimental groups. (F to J) Representative images of Sirius Red staining and IF staining (α-SMA, vimentin, and ROR-γ, Cy3-labeled, red fluorescence; n = 6; scale bar, 20 μm) of serial kidney sections from BSA nephritis mice showed reduced renal fibrosis following 2-BP treatment. (K to N) In line with these histological improvements, 2-BP administration also markedly preserved renal function, as evidenced by lower levels of urinary protein (K), urinary protein-to-creatinine ratio (L), serum creatinine (M), and blood urea nitrogen (N) in the BSA nephritis mice ( n = 6). (O to R) WB revealed that administration of 2-BP led to a marked reduction in the renal expression of ROR-γ, IL-17A, α-SMA, and vimentin in BSA nephritis mice. To ensure equal protein loading, β-actin and vinculin served as internal control proteins throughout the assays. The right panel presents semiquantitative analyses derived from the corresponding WB data, thereby providing quantitative support for the observed changes. Differences among 3 or more groups are assessed by one-way ANOVA. All results are expressed as mean ± SD. Statistical significance is denoted as follows: ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. The schematic diagram in (E) is created using BioRender.com under a valid license.

Article Snippet: Human serum IL-17 and CCL20 levels were measured using ELISA kits (R&D Systems, USA), with absorbance recorded at 450 nm and background corrected at 540 nm.

Techniques: Modification, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Control, Staining, Labeling, Fluorescence, Derivative Assay