Fig 1: ADAM9 cleaves ephrin-B1 and -B2 and can substitute for Xenopus ADAM13 in neural crest induction.A and B, HEK293T cells were transfected with plasmids encoding N-terminally HA-tagged ephrin-B1 (A) or -B2 (B) and C-terminally myc-tagged WT ADAM9 or the E348A mutant, and Western blotting was carried out using the indicated antibodies. Arrowheads point to the shed ectodomain of ephrin-Bs. C and D, HCT116 cells were transfected with the indicated siRNA, and RT-qPCR (C) and Western blotting (D) were carried out for ADAM9 mRNA and protein, respectively. Unpaired t tests were performed for log2FC of mRNA levels obtained for three biological replicates in (C), and error bars represent SEM. E-G, HCT116 (E and G) or SW620 (F) cells were transfected with the indicated siRNA and plasmid, and Western blotting was carried out using an anti-HA (E and F) or phospho-EphB3 (G) antibody. H, two-cell stage Xenopus tropicalis embryos were injected in one blastomere with the indicated MO and mRNA, allowed to develop to stage ∼12.5, and processed for in situ hybridization for snai2. χ2 test was performed for the percentage of normal embryos (n indicates total number of embryos examined). Red asterisks denote the injected side, and arrow indicates the expansion of snai2 expression domain. The scale bar represents 250 μm. CL, cell lysates; CM, conditioned media; CT, control; FC, fold change; M, mature form; P, pro-form (same below). ADAM, A Disintegrin and Metalloprotease; RT-qPCR, quantitative RT-PCR; MO, morpholino.