XB-IMG-148018
Xenbase Image ID: 148018
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Figure 2. ADAM13 Cleaves EfnB1/B2 in Cultured Cells and X. tropicalis Embryos(A) Cultured HEK293T cells were transfected with DNA constructs (0.5 μg each) expressing EfnB2 (with an N-terminal HA tag) and wild-type or the E/A mutant of each ADAM (with C-terminal myc tags). Western blots of conditioned media (CM) and whole-cell lysates (CL) were carried out using the indicated antibodies (α-HA or α-myc). Arrows indicate the ectodomain of EfnB2 shed by ADAM13; single and double arrowheads indicate the pro- and mature forms of ADAMs, respectively.(B) Conditioned media of HEK293T cells transfected to express EfnB2 and wild-type ADAM13, as in (A), were treated with or without PNGase and processed for western blot with an anti-HA antibody. The deglycosylated product is denoted with an arrow.(C) Cartoon indicating the approximate location of the ADAM13 cleavage site in EfnB2.(D and E) Each of the two anterior-dorsal blastomeres of eight-cell stage X. tropicalis embryos was injected with control (CT) or 13-1 MO (1.5 ng). Embryos were allowed to develop to stage ∼19, the heads collected by dissection, and western blots of head lysates carried out using the C-18 antibody. Relative intensity of EfnB1/B2 was determined by normalizing the western blot density of EfnB1/B2 band against that of β-actin, and by setting the amount calculated for control MO to 100%. Error bars represent SDs calculated for seven independent experiments. ∗p = 0.03. Image published in: Wei S et al. (2010) Copyright © 2010. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |