Xenbase Image ID: 137357

Figure 5. ADAM10 cleaves ephrinB2 and regulates ephrinB2 protein levels(a) ADAM10 is responsible for shedding of the ephrinB2 ectodomain. Western analysis of embryos expressing ephrinB2-HA with ADAM10-V5 and/or the ADAM10 specific inhibitor GI254023X. The full-length ephrinB2-HA and the C-terminal fragments (CTFs) of ephrinB2-HA are indicated. ADAM10-V5 is indicated, as is the Erk2 loading control.. (b) EphrinB2 associates with ADAM10. ADAM10-HA immunoprecipitation and Western analysis from embryonic lysates co-expressing ephrinB2-Flag and ADAM10-HA or ADAM17-HA. Direct lysates are probed with anti-Flag or HA as indicated. (c) ADAM10 associates with ephrinB1 as well as ephrinB2. Western analysis of the indicated IPs or direct lysates from oocytes expressing ephrinB2-HA or ephrinB1-HA alone or with ADAM10-V5. (d) ADAM10 specifically targets ephrinB2. Western analysis with indicated antibodies of lysates from embryos exogenously expressing increasing amounts of ADAM10-V5 along with ephrinB1-HA or ephrinB2-HA. (e) Endogenous ADAM10 associates with ephrinB2. EphrinB2 was immunoprecipitated from HT-29 cells and Western analysis was performed using anti-ephrinB2 or ADAM10 antibodies. EphrinB2 and ADAM10 expression levels in HT-29 cell lysates are shown. (f) In the presence of MG132, F1aMO leads to increased association between exogenously expressed ephrinB2 and ADAM10. Western analysis of the HA (ephrinB2) IPs and direct lysates from embryos co-expressing ephrinB2-HA and an ADAM10 mutant with compromised protease activity (ADAM10 PD-V5) and injected with the indicated MOs. (g) Endogenous ephrinB2 is cleaved and degraded in the presence of F1aMO, but is partially rescued by an ADAM10 specific inhibitor. Western analysis of lysates from neural folds of embryos injected with the indicated MOs, and the ADAM10 inhibitor GI254023X (st. 9 injection into the blastocoel). Westerns were probed using C-18 (pan-ephrinB) and ERK2 antibodies. (h) ephrinB2 is degraded by both the proteosomal and dynamin-dependent degradation pathways in the absence of flotillin-1. Western analysis with the indicated antibodies of embryos injected with ephrinB2-HA RNA alone or with F1aMO, and treated with vehicle control DMSO (−), or MG132 (M), and/or dynamin inhibitor dynasore (D). Erk2 is used as loading control. Image published in: Ji YJ et al. (2014) Image downloaded from an Open Access article in PubMed Central. Image reproduced on Xenbase with permission of the publisher and the copyright holder. Larger Image Printer Friendly View

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