Figure 3 | Scientific Reports

Figure 3

From: Functionally confirmed compound heterozygous ADAM17 missense loss-of-function variants cause neonatal inflammatory skin and bowel disease 1

Figure 3

(a) Effect of variants on the phorbol 12-myristate 13-acetate (PMA)-induced maturation of a disintegrin and metalloprotease 17 (ADAM17). Adam10/17 double-knockout mouse embryonic fibroblasts (Adam10/17−/− mEFs) were transfected with pFLAG-syn-hADAM17 expression constructs, incubated for 48 h and treated with PMA (100 ng/mL) in serum-free medium for 5 min. After subsequent incubation at 37 °C in DMEM for indicated times, cells were lysed in RIPA buffer supplemented with a protease inhibitor. Panels show representative results from three independent western blots analysing the expression of FLAG-tagged mature ADAM17 (mADAM17) and its preform (pADAM17) and an internal control β-actin (ACTB). The ratio of the densitometric signal of mADAM17 and pADAM17 was shown at the botttom. Full-length blots were presented in Supplementary Figure S5b. (b) Effect of variants on PMA-induced cell-surface localisation of mature ADAM17. HEK293 cells transfected with pFLAG-syn-hADAM17 expression constructs and incubated for 48 h were treated with PMA (100 ng/mL) in serum-free medium for 5 min. After incubation at 37 °C in DMEM for indicated times, cell-surface proteins were biotinylated and harvested. Biotinylated proteins precipitated using streptavidin beads were immunoblotted. The panel shows the representative result from three independent experiments. A full-length blot with a size marker image was presented in Supplementary Figure S5c. (c) Quantification of HiBiT-tagged ADAM17 localised on the cell surface. Adam10/17−/− mEFs transfected with pHiBiT-ADAM17-WT, pHiBiT-ADAM17-C567R, or pHiBiT-ADAM17-C600Y and incubated for 48 h in 96-well microplates were treated with PMA (20 ng/mL) for 2 h. Extracellular and lytic luciferase activities were measured as described in the “Materials and methods” section (Supplementary Figure S2B). Values are expressed as the ratio of extracellular or lytic luminescence intensity for each well (mean ± SD, n = 6). *P < 0.05. Data are representative of at least three experiments with similar results.

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