Fig 1: TGF-β-Enhanced Interaction of Ankyrin-G and Usp9X Regulates Spine Morphogenesis(A) SIM image of mCherry-expressing neurons (red) with treatment of vehicle or TGF-β (20 ng/mL) for 1 h to detect the interaction between ankyrin-G and Usp9X by PLA (cyan). Scale bar, 10 μm, top. Magnified image of boxed spine and dendrite (bottom). Scale bar, 2 μm, bottom.(B) The number of PLA puncta per 10 μm in neurons treated with vehicle or TGF-β. Bars also show the distribution of PLA puncta in spines (white) and dendrites (gray) (vehicle, n = 28 neurons; TGF-β, n = 33 neurons). **p < 0.01; ***p < 0.001 by two-tailed unpaired t test.(C) Confocal images of neurons transfected with control RNAi (control) or shRNA against Usp9X (shUsp9X) or ankyrin-G (shAnkG) and subsequently treated with vehicle (top) or TGF-β (20 ng/mL) (bottom) for 1 h. Scale bar, 5 μm.(D and E) Spine head area (D) and density (E) in control (black bars), shUsp9X (red bars), and shAnkG (blue bars) with vehicle (plain pattern) or TGF-β (comb pattern) (n = 20 neurons for each group). *p < 0.05; ***p < 0.001; †††p < 0.001 by one-way ANOVA followed by non-parametric statistical analysis. All data are presented as mean ± SEM.
Fig 2: TGF-β-Mediated Phosphorylation of Usp9X Enhances the Interaction with Ankyrin-G(A) Usp9X homology model (gray) was generated using crystal structure (PDB: 5WCH) as template. S1,593, S1,600, and S1,609 (orange) are positioned on a loop absent from crystal structure 5WCH. Energy minimization of this loop allowed an energetically favorable conformation to be predicted (blue) and suggests that all serine sites lie distal to the catalytic triad (magenta).(B) Representative western blot of His-Usp9X1,547–1,962, expressed and purified from E. coli, was used to pull down HEK293T-expressed hemagglutinin (HA)-AnkG1–808 by nickel affinity purification. A GFP-transfected sample was used as a negative control.(C) Representative western blot of HA-AnkG1–807 with co-expressing FLAG-Usp9X1,555–1,958 (FLAG-Usp9XWt) or FLAG-Usp9X1,555–1,958 (S1,593;1,600;1,609A) (FLAG-Usp9XS3A) construct from HEK293T cells. Immunoprecipitation (IP) of phosphoserine (pSer) and detection of phosphorylated Smad2 (p-Smad2) were performed to confirm TGF-β-mediated signaling.(D) In situ PLA measurement of the interaction between HA-AnkG1–807 and FLAG-Usp9XWt or FLAG-Usp9XS3A in HEK293T cells. Scale bar, 5 μm.(E) Quantification of PLA signal from (D) (n = 9–16 cells). HA-AnkG1–807 and FLAG-Usp9XWt (black bars) or FLAG-Usp9XS3A (red bars) with vehicle (plain pattern) or TGF-β (comb pattern). ***p < 0.001; †††p < 0.001 by two-way ANOVA followed by Bonferroni post-tests. All data are presented as mean ± SEM. TGF-β (−), vehicle; TGF-β (+), 20 ng/mL TGF-β for 1 h.See also Figure S1.
Fig 3: TGF-β-Mediated Phosphorylation of Usp9X Regulates Spine Morphogenesis through Interaction with Ankyrin-G(A and B) SIM images of a spine (A) from FLAG-Usp9XWt in Figure 4C. Lower panels show ratiometric images and colocalization (white). Scale bar, 0.5 μm. 3D reconstruction (B) of a PLA-SIM image of a spine from TGF-β (+). Scale bar, 1 μm.(C–F) Correlation plots of the area of FLAG-Usp9X versus HA-AnkG nanodomains (C) and the area of FLAG-Usp9X (D) or HA-AnkG (E) nanodomains versus the area of PLA nanodomains and spine head area versus the area of PLA nanodomains (F) from the HA-AnkG + FLAG-Usp9XWt + TGF-β(−) group or the HA-AnkG + FLAG-Usp9XWt + TGF-β(+) group. Two-tailed non-parametric Spearman correlation was performed, and the 90% confidence interval (CI) was considered for equivalence tests. TGF-β (−): vehicle; TGF-β (+): 20 ng/mL TGF-β for 1 h.
Fig 4: TGF-β-Mediated Phosphorylation of Usp9X in Dendrites Is Required for the Development of Spines(A) Confocal images of primary cortical neurons for detection of interaction between the whole construct of HA-AnkG and FLAG-Usp9XWt or FLAG-Usp9XS3A with PLA. Scale bar, 20 μm.(B) Bar graph of PLA signal with vehicle (plain pattern) or TGF-β (comb pattern) treatment in HA-AnkG and FLAG-Usp9XWt (black bars) or FLAG-Usp9XS3A (red bars) expressing cells (n = 16 neurons for each group). ***p < 0.001; †††p < 0.001 by two-way ANOVA followed by Bonferroni post-tests. All data are presented as mean ± SEM.(C) In situ PLA/SIM images of dendritic regions in neurons from (A). Scale bar, 5 μm.(D–F) Spine head size in the presence or absence of ankyrin-G (D) and with vehicle (plain pattern) or TGF-β (comb pattern) treatment (F); theratio of spines expression HA-AnkG and PLA signal (E) (n = 16 neurons for each group). ***p < 0.001; †††p < 0.001 by two-tailed unpaired t test (D) or one-way ANOVA followed by non-parametric statistical analysis (F). All data are presented as mean ± SEM.See also Figures S2 and S3.
Supplier Page from OriGene Technologies for Usp9x Mouse shRNA Plasmid (Locus ID 22284)