Figure 4

Figure 4

The impact of human A20 mutants on A20^{∆127/∆127} zebrafish survival. (A) Survival data for A20^+/+, A20^+/∆127 and A20^{∆127/∆127} zebrafish at 3 wpf (zfA20), mock injected (Sham) or with ectopic administration of wild-type human A20 (hA20^WT), C103A, S381A or C243Y mutants. Numbers indicate number of genotyped survivors expressing the respective human gene variant. Each box indicates the survivors from an independent experiment. Survival frequency data is represented in the pie chart. N indicates the total number of zebrafish embryos injected for each A20 mutant. Statistical difference was observed between zfA20 and C103A injected/rescued fish (*p < 0.05) and between C103A and C243Y injected fish (*p < 0.05). (B) NF-κB luciferase assay for HEK293 cells transiently non-transfected (NTC), or co-transfected with wild-type human A20 (hA20^WT) or the C103A, S381A or C243Y mutant and treated with or without hTNFα. Data represented as mean ± SD. P-values were determined using ANOVA. Each dot represents an individual experiment and are representative of N = 3–5 experiments per A20 mutant. Below is shown a representative Western blot for each A20 mutant. The displayed gel has been cropped to improve clarity and conciseness of the figure. Software used for this Figure: GraphPad Prism v7.0 (https://www.graphpad.com/scientific-software/prism/), Adobe Illustrator CC 2018 (https://www.adobe.com/au/products/illustrator.html), Bio-rad ChemiDoc v6 (https://www.bio-rad.com/en-au/product/chemidoc-imaging-system?ID=OI91XQ15).