Xenbase Image ID: 173304

Fig. 5. The cytoplasmic K911 residue is crucial for ubiquitin-proteasome-mediated ADAM13 turnover and regulation by ADAM19. (A,F) Responses of different variants of ADAM13 to ADAM19 KD. Embryos were injected in one blastomere at the two-cell stage with the indicated RNA (100 pg each) and MO (6 ng each), and cultured to stage ∼19. Whole-embryo lysates were processed for western blot for the C-terminal myc6 tag on the ADAM constructs. A shorter exposure was performed for the ΔC mutant in B,E. Ubiquitylation of wild-type ADAM13 and the K911R mutant. HEK293T cells were transfected to express the FLAG-tagged ubiquitin with and without HA-tagged ADAM13 (B), or myc6-tagged wild-type ADAM13 or the K911R mutant (E). Immunoprecipitation and western blot were carried out with the indicated antibodies. (C) MG132 protects ADAM13 and rescues the effect of ADAM19 KD on ADAM13. Embryos were injected, dissociated and cultured in the absence or presence of MG132. Cell lysates were processed for western blot. (D) XTC cells were transfected with 0.5 µg of plasmids encoding myc6-tagged wild-type ADAM13 or the K911R mutant. Immunocytochemistry for myc (green) and DAPI labeling for nuclei (blue) with images taken in green and blue channels merged. Single and double arrowheads indicate the pro- and mature forms of ADAM13, respectively; asterisk denotes the autocleavage product. UB, ubiquitin. Image published in: Li J et al. (2018) Copyright © 2018. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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