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. 2014 Dec 10;34(50):16637–16649. doi: 10.1523/JNEUROSCI.2452-14.2014

Figure 5.

Figure 5.

Synaptic activity levels regulate Nedd4-1 and USP8 in opposing fashion. A, Ubiquitination of AMPARs after chronic bicuculline treatment. Cultured cortical neurons 18–22 DIV were treated with bicuculline (20 μm) for 24 h before IP with GluA2/3 antibodies in nondenaturing IP conditions. IPs were resolved by SDS-PAGE and probed with anti-ubiquitin, anti-GluA1, and anti-GluA2/3 antibodies. B, Chronic bicuculline-induced changes in Nedd4-1 and USP8 protein levels. Representative Western blots from total lysate of cortical neurons treated with bicuculline (20 μm) for 72 h compared with untreated neurons. Cell lysates were resolved by SDS-PAGE and probed anti-GluA1, anti-Nedd4-1, anti-USP8, and anti-tubulin antibodies. C, Quantification of immunoblots for total GluA1, Nedd4-1, and USP8 levels normalized to tubulin and control condition, n ≥ 8 samples per condition. D, E, Chronic application of bicuculline promotes the redistribution of Nedd4-1. Dissociated hippocampal neurons 18–22 DIV expressing GFP HA-Nedd4-1 (Sindbis, 24 h) were treated with bicuculline (20 μm, 24 h) or left untreated and stained with anti-HA (red) and anti-GFP (green) antibodies. Redistribution was quantified using local maxima macro; n ≥ 20 dendrites per condition over 2 independent experiments (D); representative maximum z-projected confocal images of whole cell and dendrite are depicted (E). Scale bar, 10 and 5 μm for whole cell and dendrite images, respectively. *p < 0.05, **p < 0.01, ***p < 0.001; Student's t test. Graphs show mean ± SEM.