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

Figure 2.

Figure 2.

Nedd4-1's C2 domain regulates its trafficking and function in neurons. A, Representative immunofluorescent images of dissociated hippocampal neurons 19–22 DIV expressing HA-tagged Nedd4-1ΔC2 (Sindbis) for 18–20 h and treated with AMPA (10 μm, 10 min) or left untreated and stained for HA (red) and GFP (green). Representative maximum z-projected confocal images of whole cell and dendrite are depicted. Scale bar, 10 and 5 μm for whole cell and dendrite images, respectively. B, Quantification of images from HA-Nedd4-1 wild-type and ΔC2-expressing cells with and without AMPA (10 μm, 10 min); ***p < 0.001, Student's t test; n > 18 dendrites per condition over 3 independent experiments. C, Representative traces of mEPSCs recorded from dissociated hippocampal neurons 18–24 DIV expressing control GFP, HA-Nedd4-1 wild-type, or HA-Nedd4-1ΔC2 (Sindbis) for 18–22 h. Scale bar, 500 ms and 20 pA. D, Cumulative probability distributions of amplitudes of all mEPSCs recorded from neurons infected with Sindbis GFP (control), HA-Nedd4-1 wild-type, or HA-Nedd4-1ΔC2. n = 2739, 1781, and 2426 events, respectively. Inset displays averaged waveform of all events in each condition. Scale bar represents 5 ms, 5 pA. E, Mean mEPSC amplitude. *p < 0.05, ANOVA with Tukey's post hoc analysis; n = 20–25 cells; data collected across 3 independent experiments. F, Representative immunofluorescent images of hippocampal neurons 18–24 DIV expressing GFP (control), HA-Nedd4-1 wild-type, or HA-Nedd4-1ΔC2 for 18–22 h and stained for surface GluA1-containing AMPARs (red) and GFP (green). Scale bar, 5 μm. G, Quantification of surface GluA1 signal in straightened dendrites of GFP-positive cells in all three conditions. *p < 0.05, ANOVA with Tukey's post hoc analysis; n = 57–59 cells; data collected across 3 independent experiments. H, Immunoblots showing reduced coimmunoprecipitation between GFP-tagged GluA1 and HA-tagged Nedd4-1ΔC2 compared with HA-tagged Nedd4-1 WT. HEK293 cells were cotransfected with GFP-GluA1 and either HA-Nedd4-1ΔC2 or HA-Nedd4-1 WT for 18–24 h. HA-Nedd4-1 was immunoprecipitated with anti-HA antibody and amount of coprecipitating GluA1 was determined through anti-GFP blotting. Graphs show mean ± SEM.