Extended Data Fig. 4: Development of specific small molecule targets ADAM9 protein.
(A) (Left) SDS-PAGE and (Right) Western blot analysis of Ni-NTA column purified recombinant ADAM9. Protein was expressed in ExpiCHO cells using the standard protocol of the GIBCO ExpiCHO Expression System. The purity of the final preparations was evaluated on Coomassie-stained SDS–PAGE gels and probed using HRP anti-His tag antibody. The arrow indicates the ADAM9 protein band which has a molecular weight of 54.4 kDa. M, molecular weight Marker; Lane 1, supernatant culture medium; Lane 2, Ni-NTA column flowthrough; Lane 3, washed 20 mM imidazole; Lane 4-5, eluted purified ADAM9. (B) The sensorgram result of Biacore SPR analysis. (C and D) Structural similarity of ADAM9 with ADAMs (C) and MMPs (D) was listed. Members with higher identity on the zinc-binding motif were marked. (E to H) Inhibition assays of ADAM9i with ADAM8 (E), ADAM12 (F), MMP1 (G), and MMP7 (H) were performed by using the recombinant human metalloproteinase and their fluorescent substrates in vitro. Mean ± SD of three independent experiments (ADAM8, ADAM12, and MMP1, n = 3 repeats). Significance was determined by one-way ANOVA in comparison with the vehicle (MMP1).
