Figure 2

(a,b) mRNA and protein expression of exogenously transfected a disintegrin and metalloprotease 17 (ADAM17) in Adam10/17 double-knockout mouse embryonic fibroblasts (Adam10/17^−/− mEFs). (a) Adam10/17^−/− mEFs were transfected with FLAG-syn-hADAM17-WT, FLAG-syn-hADAM17-C567R, or FLAG-syn-hADAM17-C600Y and incubated for 48 h. Amounts of exogenous ADAM17 mRNA were measured by qRT-PCR, using GAPDH mRNA as an endogenous control. Values are expressed as fold changes (mean ± SD, n = 3) compared with respective values in cells transfected with syn-hADAM17-WT. (b) Transfected cells were lysed in M-PER buffer supplemented with a protease inhibitor. Panels show representative results from three independent western blots analysing the expression of FLAG-tagged ADAM17 and β-actin. Full-length blots were presented in Supplementary Figure 5a (c,d) Phorbol 12-myristate 13-acetate (PMA)-stimulated shedding activity for various substrates in Adam10/17^−/− mEFs exogenously transfected with ADAM17. (c) Adam10/17^−/− mEFs were co-transfected with pFLAG-syn-hADAM17 expression constructs and AP-tagged TNF-α, TGF-α, or HB-EGF expression constructs. Relative AP activity released into the media of transfected Adam10/17^−/− mEFs during the 2-h stimulation with PMA (20 ng/mL) in the presence or absence of the metalloprotease inhibitor BB94 (1 μM). Data represent three independent experiments. Error bars indicate mean ± SD. *P < 0.05. (d) Adam10/17^−/− mEFs were co-transfected with pFLAG-syn-hADAM17 expression constructs and AP-tagged TNF-α or TGF-α expression constructs. Relative AP activity released into the media of transfected Adam10/17^−/−mEFs during the 2-h stimulation with PMA (20 ng/mL) in the presence or absence of the human ADAM17 inhibitory antibody (15 μg/mL). Data represent three independent experiments. Error bars indicate mean ± SD. *P < 0.05.