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. 2019 Mar 4;11(4):e9695. doi: 10.15252/emmm.201809695

Figure 5. Neuronal stimulation by NMDA increases ADAM10‐mediated NrCAM shedding.

Figure 5

  • A
    To ensure proper synapse formation, primary neurons were kept in culture for 10 days prior to the treatment (Wan et al, 2012). Then, cells were treated with NMDA (50 μM), NMDA (50 μM) and GI254023x (5 μM), GI254023x (5 μM) alone or vehicle for 30 min. Total cellular mADAM10 levels remained unchanged by the treatment, which is in line with NMDA altering mainly the intracellular localization of ADAM10 by driving the protein to the synaptic membranes (Marcello et al, 2007). Densitometric quantifications of the Western blots are shown. One‐way ANOVA with post hoc Dunnett's test (***P < 0.001, n = 7). Given are mean ± the standard error of the mean. The mean levels of solvent‐treated cells were set to 1. The membrane was reprobed with the different indicated antibodies.
  • B
    Wt neurons were kept in culture until DIV10; then, the cells were treated with NMDA (50 μM), or vehicle for 30 min. Cell surface proteins were labeled with biotin and enriched by streptavidin pull‐down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAMs surface/lysate levels. Densitometric quantifications of the Western blots are shown. Two‐sided Student's t‐test (**P < 0.01, n = 6). Given are mean ± the standard error of the mean. The mean levels of solvent‐treated cells were set to 1. Representative Western blots are shown.

Source data are available online for this figure.