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. 2017 Apr 25;292(23):9451–9464. doi: 10.1074/jbc.M116.772442

Figure 2.

Figure 2.

STRN4 expression at excitatory synapses depends on spontaneous NMDA receptor activity. A, Western blot analysis showing that the expression of STRN4 protein increased along maturation of hippocampal neurons. The intensity of STRN4 was normalized with that of actin. Results were pooled from three independent experiments. B, developmental up-regulation of STRN4 expression at 14 DIV was blocked by TTX (2 μm). Results were pooled from four independent experiments. C, hippocampal neurons (16 DIV) were treated with vehicle (Con), the NMDA receptor antagonist APV (100 μm), or the AMPA and kainate receptor antagonist DNQX (20 μm) for 48 h, and STRN4 protein expression was examined by Western blotting. Results were pooled from four independent experiments. D, Strn4 mRNA level was examined in dissociated rat hippocampal neurons (18 DIV) by in situ hybridization following treatment with vehicle (control) or APV (100 μm) for 48 h. The number of Strn4 mRNA (cyan) granules in the cell soma or dendrites was not significantly different between the two conditions (n = 17); p > 0.05, Student's t test. E, representative images of GFP-transfected hippocampal neurons treated with vehicle (control) or APV (100 μm) at 16 DIV for 48 h, followed by immunofluorescence staining using STRN4 antibody. STRN4 intensity (cyan or gray) in dendritic protrusions (arrowheads) and cell soma (insets) were quantified. APV treatment specifically reduced STRN4 fluorescence intensity in dendritic protrusions but not the cell soma (15 dendrites from 7 neurons were quantified for each condition). F, double immunofluorescence staining of GFP-transfected hippocampal neurons by STRN4 and PSD-95 antibodies indicated that the synaptic STRN4 was significantly reduced by APV (100 μm for 48 h) in dendritic spines containing PSD-95 clusters (arrowheads). (9 dendrites from 6 to 7 neurons were quantified for each condition.) Data are mean ± S.E. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; Student's t test. Scale bars, 20 μm (upper panels in D and E) or 5 μm (lower panels in D–F).