Journal: Cell Chemical Biology

Article Title: Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET

doi: 10.1016/j.chembiol.2021.05.002

Figure Lengend Snippet: The effect of N-terminal NanoLuciferase, HaloTag, or SNAP-tag additions to IL-23 receptor subunits on IL-23-induced STAT3 phosphorylation STAT3 phosphorylation induced in HEK293T cells transfected with different tagged variants of the IL-23 receptor after a 30-min incubation with increasing concentrations of IL-23. Data are expressed as a percentage of the response obtained with 15 nM IL-23. Values are mean ± SEM from four or three (NL-IL23R and HT-IL12Rβ1) independent experiments.

Article Snippet: AlphaLISA SureFire Ultra p-STAT3 (Tyr705) Assay Kit , Perkin Elmer , Cat# ALSU-PST3.

Techniques: Transfection, Incubation

Journal: Cell Chemical Biology

Article Title: Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET

doi: 10.1016/j.chembiol.2021.05.002

Figure Lengend Snippet:

Article Snippet: AlphaLISA SureFire Ultra p-STAT3 (Tyr705) Assay Kit , Perkin Elmer , Cat# ALSU-PST3.

Techniques: Recombinant, Modification, Staining, Purification, Luciferase, Expressing, Plasmid Preparation, Software

Journal: Nature Communications

Article Title: Characterisation of IL-23 receptor antagonists and disease relevant mutants using fluorescent probes

doi: 10.1038/s41467-023-38541-2

Figure Lengend Snippet: a Left: Crystal structure of N -terminal domains 1–3 of IL23R (orange) in complex with IL-23 (IL23p19 = blue, IL12p40 = green) and the N -terminal domain of IL12Rβ1 (PDB: 6WDQ ). Right: The interface between IL23R and IL23p19 with C115 coloured cyan. b The phospho-STAT3 signal generated when cells expressing different constructs were treated with 5 nM IL-23. Data are mean ± SEM from five or six (untagged) or three (NanoLuc tagged) independent experiments conducted in triplicate. For the IL23R + IL12Rβ1 data set, one outlier (11108.7) was detected using the Grubbs test (with alpha = 0.001) and removed from the statistical analysis. Statistical significance of the C115Y mutation on the alphaLISA data was assessed using a 2-way ANOVA with Tukey’s multiple comparison test. *** p < 0.001. The p value for the comparison of NL tagged construct response was 0.0001 and the p value for the comparison of untagged receptor was 0.951. c Luminescence signal measured when IL12Rβ1 was expressed with either NL-IL23R or the C115Y mutant. Data are mean ± SEM generated from the mean luminescence values of seven (C115Y) or eight independent experiments performed in pentuplicate. d The proportion of luminescence from c that emanated from extracellular protein (as defined by the reduction in luminescence after treatment with 60 μM NanoLuc extracellular inhibitor). Data are mean ± SEM generated from the mean luminescence values of five (C115Y) or six independent experiments performed in triplicate. e The BRET ratio generated from HaloTag-618 labelled cells expressing NL tagged wildtype or C115Y IL23R with HT-IL12Rβ1 or unlabelled IL12Rβ1. Data are mean ± SEM generated from the mean BRET values of three independent experiments performed in pentuplicate. Statistical significance of differences in mean values shown in c – e were measured using a 2-sided, non-paired t test with ** indicating a p value of <0.005 and *** indicating a p value of <0.0005. The p values for the results shown in c , d and e were p = 0.148, 0.0005 and 0.0013 respectively. Source data are provided as a Source Data file.

Article Snippet: The following day media was replaced with serum free DMEM and the cells incubated for 3 h. Media was then replaced with HBSS containing 1 mg/ml BSA and increasing concentrations of IL-23 and the cells incubated for 30 min. An AlphaLISA SureFire Ultra assay kit (Perkin Elmer #ALSU-PST3) was then used to measure STAT3 phosphorylation at residue Tyr705.

Techniques: Generated, Expressing, Construct, Mutagenesis, Comparison