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. 2016 May 19;7:11613. doi: 10.1038/ncomms11613

Figure 2. Copine-6 accumulates in spines on NMDA-mediated calcium influx.

Figure 2

(a) Subcellular fractionation of adult rat brain in the presence of calcium (top) or EDTA, added after the first centrifugation step (bottom). P1–P3, pellets; PSD, postsynaptic densities; S1–S6, supernatants; SM, synaptic membranes (see Methods for details). (b) Representative pictures (left) and quantification (right) of DIV21 cultured hippocampal neurons transfected at DIV14 to express Copine-6-GFP and tdRFP after the indicated treatment. For quantification, the ratio of Copine-6-GFP fluorescence in the spine to that in the dendrite was normalized to spine volume (see Methods for details). Copine-6-GFP becomes enriched in spines after 5 min of cLTP (cLTP 5′). The accumulation of Copine-6-GFP is lost after an additional 40 min in ACSF (cLTP 5′+40′ ACSF). Data are mean±s.e.m. from n=500 spines per condition (25 spines per neuron from 20 neurons of each condition; from 3 independent cultures). ***P<0.001 by one-way ANOVA with Tukey's post-hoc test. Scale bar, 2 μm. (c) Time-lapse confocal pictures of DIV14 hippocampal neurons expressing Copine-6-GFP (green) and cytosolic tdRFP (red) during stimulation with 10 μM NMDA. Time points indicate minutes after NMDA application. Scale bar, 20 μm.