Xenbase Image ID: 137358

Figure 6. ephrinB2 protein half-life is decreased in the absence of flotillin-1 and partially rescued by the ADAM10 inhibitor(a) Two-cell stage embryos were injected with carefully titrated ephrinB2-HA RNA along with control MO or F1aMO to yield roughly equivalent ephrinB2 protein levels when embryos reached the early neurula stages. A group of the injected embryos was subjected to a secondary injection into the blastocoel at stage 9 with ADAM10 inhibitor (10 nl of 1 mM), and the archenteron cavity (stage 14) with cycloheximide (10 nl of 75 ug/ul), and externally incubated in cycloheximide (7.5 ug/ul) to block further protein synthesis for the indicated times. Western blot analysis was performed on the embryonic lysates using HA antibodies, or Erk2 antibodies (as a loading control). (b) A graph of the mean band intensities as measured by Image J software shows the approximate half-lives in the presence of cycloheximide and the indicated MOs and ADAM10 inhibitor. The ephrinB2 C-terminal fragments (short arrow) and the full length protein (long arrow) are indicated. These data are the result of three independent experiments and +/− represents sd. (c) Endogenous surface ephrinB2 levels are reduced by knockdown of flotillin-1, but prominently rescued by the ADAM10 inhibitor. Embryos were injected with the indicated MOs and inhibitors. Neural folds were excised and left non-biotinylated (lane 1) or biotinylated. Lysates were prepared and cell surface proteins immunoprecipitated with streptavidin conjugated sepharose. Western analysis of biotin labelled cell surface proteins was performed using anti ephrinB antibody. Direct Western analysis of neural fold lysates were probed for ephrinB. Erk2 expression is shown as a 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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