Fig. 3.

The highly conserved motif within the IRHD is crucial for ADAM17 maturation and activity in iRhom deficient cells. MEFs, deficient for iRhom1 and iRhom2, stably expressing the indicated iRhom constructs were used for the described experiments. As negative control, cells stably expressing GFP (ctr) were used. a–b To analyse the maturation of ADAM17, glycosylated proteins were enriched by concanavalin A beads (ConA) and immunoblotted. The transferrin receptor (TfR) served as input control. To analyse the binding between ADAM17 and iRhom constructs, co-IP were performed by using the iRhom constructs (with HA tag) as bait. Quantitative analysis of ADAM17 binding can be found in figures S5b, c. n > 3. c Relative amount of imADAM17 from conA precipitations was assessed by densitometric measurements from immunoblots (a) and (b). The ratio between imADAM17 and TfR was calculated and normalised to wt miRhom2. n > 5. d–f ADAM17-mediated shedding activity was assessed by an AP assay in iRhom1/iRhom2-deficient MEFs. The ADAM17 substrates transforming growth factor (TGFα) (d), amphiregulin (AREG) (e) and heparin-binding EGF-like growth factor (HBEGF) (f) each tagged with AP were utilised. Cells were incubated for 2 h under unstimulated condition, stimulated with the phorbol ester PMA (phorbol-12-myristate-13-acetate) or stimulated with PMA and treated with the inhibitor TAPI1. n = 4