FIGURE 9.
Knocking down endogenous RNF34 in cultured hippocampal neurons increases both the γ2-GABAAR cluster density and GABAergic innervation. A, RNF34 shRNAs (Sh1 and Sh2) used in this study. The three point mutations in Sh1 3m and Sh2 3m are shown in red. B, Sh1 and Sh2 significantly knocked down the protein expression of HA-RNF34 (in pRK5 plasmid), compared with HEK293 cells co-transfected with HA-RNF34 and various control plasmids (mU6, Sh1 3m, or Sh2 3m). Cell lysates were immunoblotted with Ms anti-HA and Ms anti-actin mAbs. C, Sh2 did not knock down the protein expression of the rescue mRNA, although Sh2 knocked down the protein expression of the HA-RNF34. D and E, representative immunofluorescence images of dendrites from hippocampal neurons that were nontransfected (NT) or co-transfected with EGFP and mU6 pro vector (mU6), or various Sh2 constructs (Sh2 or Sh2 3m), or Sh2 + rescue. Immunofluorescence with Rb anti-RNF34 (red in D), Rb anti-γ2 (red in E) or sheep anti-GAD (blue in E). Dendrites of transfected neurons were identified by EGFP fluorescence (green). Scale bar, 5 μm. F–H, quantification of the effect of knocking down RNF34 with Sh2 on the RNF34 fluorescence intensity (F), γ2-GABAAR cluster density (G), and GAD boutons contacting the transfected neurons (H). *, p < 0.05; ***, p < 0.001 in one-way ANOVA Tukey-Kramer multiple comparison test, n = 16 neurons for each group from four independent transfection experiments.