Xenbase Image ID: 159150

Figure 1. adam13 cleaves pcdh8l.(A) Schematic representation of full length pcdh8l. A Flag tag (DYKDDDDK) was introduced just before the first cadherin repeat (EC1). The yellow arrow indicates the predicted cleavage site of adam13 based on the molecular weight of N- and C- terminal fragments. Red and blue asterisk indicate N and O glycosylation site, respectively. The monoclonal antibody 2F4 was produced against the cytoplasmic domain (Red). (B) Western blot from transfected Hek293T cells. Glycoproteins from the conditioned media were purified on concanavalin-A agarose. The cleavage fragment was detected using the N-terminus Flag tag with the mAb M2. Total pcdh8l and adam13 were detected using mAb 2F4 and mAb 4A7, respectively. The Pro (P) and Metalloprotease (M) active forms of adam13 are indicated. (C) Histogram representing the percentage of embryos lacking fluorescent neural crest cell in the migration pathway. (D) Photographs of representative embryos with or without fluorescent migrating neural crest cells. Embryos were injected at the one-cell stage with a morpholino targeting adam13 (10 ng MO13). Messenger RNA for RFP alone or combined with the different constructs was injected at the 8-cell-stage in a dorsal animal blastomere. Each injection was compared to RFP injected in control embryos in which RFP positive cranial neural crest cells have successfully migrated into the branchial and hyoid arches (0% inhibition). N = number of embryos scored from three or more independent experiments. Error bars represent standard error to the mean. One-way ANOVA was performed to determine statistical significance. Statistically significant at *p<0.05, ***p<0.005.10.7554/eLife.26898.005Figure 1—source data 1. Source data for Figure 1.Figure 1—figure supplement 1. Characterization of mAb 2F4 to pcdh8l.(A) Western blot from total membrane extracted from 10 embryos at gastrula stage (Stage 10) and early tailbud stage (Stage 20) detected using mAb2F4. (B–C) Cranial neural crest explants on fibronectin. Explants were left to migrate for 5 hr and fixed in 1XMBS, 3.7% formaldehyde for 1 hr, permeabilized for 30 min in 1XMBS containing 0.5% TritonX100. Staining with mAb 2F4 (B) clearly shows a membrane staining while DAPI stains the nuclei (C). (D) Wholemount immunostaining of tailbud stage embryo fixed in DENTS (20% DMSO 80% Methanol) using mAb 2F4. The red arrows indicate the tip of the cranial neural crest segments. The staining is identical to the one obtained by in situ hybridization with the pcdh8l probe. (E) Western blot on Hek293T cells extract transfected with the various Xenopus laevis protocadherin. The mAb 2F4 only recognizes pcdh8l (PCNS). Larger Image Printer Friendly View

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