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. Author manuscript; available in PMC: 2021 Feb 5.
Published in final edited form as: Neuron. 2019 Dec 5;105(3):506–521.e7. doi: 10.1016/j.neuron.2019.11.003

Figure 3.

Figure 3.

Usp9X regulates spine morphology by deubiquitination of ankyrin-G. (A) Confocal images of neurons expressing scramble (control RNAi) or Usp9X knockdown (shUsp9X) construct. Scale bar = 40 μm for the left panel and 5 μm for the right panel. (B, C) Bar graph showing shUsp9X causes a decrease in spine head size and density compared with control. Control from 9 cells and shUsp9X from 10 cells were analyzed; **p = 0.0025, *p = 0.0114. Two-tailed unpaired t-test was performed. (D) Confocal images of control RNAi or shUsp9X transfecting neurons co-expressing GFP-Usp9X1555-1958 construct. Scale bar, 5 μm. (E, F) Bar graph of spine head size and density showing co-expression of GFP-Usp9X1555-1958 with control or shUsp9X. 20 neurons were analyzed from each group. ***p < 0.001, †p < 0.05; Two-way ANOVA was followed by Bonferroni post-tests. (G) The peptidase domain of Usp9X (Flag-Usp9X1555-1958) promotes ankyrin-G deubiquitination. Cell lysates were analyzed from HEK293T cells. (H) Confocal images of neurons expressing control RNAi or shUsp9X, co-expressing GFP-ankyrin-G or GFP-ankyrin-G-Muta;b. Scale bar, 5 μm. (I, J) Quantification of the effects on spine size or density. 20 neurons were analyzed per each group. **p < 0.01, ***p < 0.001; One-way ANOVA was followed by Bonferroni post-tests. Data are represented as mean ± SEM. See also Figure S3.