FIGURE 8.
Cultured hippocampal neurons overexpressing RNF34 show reduced density of endogenous γ2-GABAAR clusters and reduced GABAergic innervations. A and B, representative immunofluorescence images of dendrites from hippocampal neurons that were nontransfected (NT) or transfected with EGFP and vector control (HA) or EGFP and various RNF34 constructs (HA-RNF34, HA-RNF34 ΔC, or HA-RNF34 H351A, all in pCAGGS plasmid). Primary antibodies were Ms anti-HA (red) in A and B, Rb anti-γ2 (blue in A), or sheep anti-GAD (blue in B). Neurons transfected with the RNF34 constructs or HA were identified by anti-HA fluorescence (red), which coincided with the fluorescence of the co-transfected EGFP (not shown for economy of space). The HA-RNF34 constructs were made in pCAGGS plasmid, and overexpression results were determined 2 days after transfection (13 DIV). Scale bar, 5 μm. C and D, quantification of the effect of RNF34 constructs on the density of γ2 clusters and the number of GAD boutons innervating the transfected neurons. Data are presented as mean ± S.E., **, p < 0.01; ***, p < 0.001 in one-way ANOVA Tukey-Kramer multiple comparison test, n = 16 neurons for each group from four independent transfection experiments.