peptide flag sfb tagged usp7 construct  (Addgene inc)

Journal: Cell reports

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1016/j.celrep.2025.115231

Figure Lengend Snippet: (A and B) Immunofluorescence of USP7 in the cerebral cortex on embryonic day 14 (E14; A) and post-natal day 42 (P42; B). Dashed lines demarcate the cortical plate (c.p.). Scale bars, 100 μm. (C) Virtual optomotor system (VOS) test: spatial frequency threshold. ** p < 0.01 by Bonferroni’s multiple-comparisons test. (D) Motor strength test: time to grip onto the inverted screen. * p < 0.05 by Dunn’s multiple-comparisons test. (E and F) CatWalk: relative mean intensity distributed onto front- and hindpaws (E) and limb swing speed (F). ** p < 0.01, *** p < 0.001 by Tukey’s multiple-comparisons test. See also . (G–I) Fear conditioning: percentage of freezing time in tone/shock pairing (G), contextual conditioning (H), and auditory cued conditioning (I). * p < 0.05, ** p < 0.01 by Bonferroni’s multiple-comparisons test ( Usp7 WT vs. cKO). (J) Representative biting injury on mice co-caged with Usp7 cKO mice. See also . (K) Percentage of tail biters among Usp7 cKO mice from 3 to 12 weeks old. (L) Schematic of behavioral phenotypes in Usp7 cKO mice that are relevant to HAFOUS. Data are presented as mean ± SEM. See also .

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, an S protein-Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Immunofluorescence

Journal: Cell reports

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1016/j.celrep.2025.115231

Figure Lengend Snippet: (A) Immunofluorescence of c-cas-3 in the cerebral cortex at different ages. Scale bar, 200 μm. (B) Schematic of the USP7-Mdm2-p53 signaling pathway regulating apoptosis. (C) Immunofluorescence of c-cas-3 in the cerebral cortex of mice of different Trp53 and Usp7 genotypes at P0. Scale bar, 200 μm. (D) Quantification of c-cas-3 fluorescent signal as in (C). * p < 0.05 by Tukey’s multiple-comparisons test. (E) VOS test: spatial frequency threshold. No significant genotype effect by one-way ANOVA. (F) Motor strength test: time to grip onto the inverted screen. * p < 0.05, ** p < 0.01 by Dunn’s multiple-comparisons test. (G and H) CatWalk: relative mean intensity distributed onto front- and hindpaws (G) and limb swing speed (H). * p < 0.05, *** p < 0.001, **** p < 0.0001 by Tukey’s multiple-comparison test. See also . (I–K) Fear conditioning: percentage of freezing time in tone/shock pairing (I), contextual conditioning (J), and auditory cued conditioning (K). * p < 0.05 by Bonferroni’s multiple-comparisons test ( Usp7 WT vs. cKO). (L) Representative biting injury on mice co-caged with Usp7 cKO; Trp53 +/− mice. (M) Percentage of tail biters among Usp7 cKO; Trp53 +/− mice from 3 to 12 weeks old. Data are presented as mean ± SEM. See also and .

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, an S protein-Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Immunofluorescence, Comparison

Journal: Cell reports

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1016/j.celrep.2025.115231

Figure Lengend Snippet: (A) Flowchart to characterize the proteome of the cerebral cortex with 10-plex TMT-MS. (B) Volcano plot showing differentially expressed proteins in TMT-MS. The p values were calculated by 2-tailed unpaired t test with Benjamini–Hochberg correction. (C) Network of GO gene sets de-enriched in Usp7 cKO cortices based on TMT protein abundance. Nodes represent gene sets (q < 0.01), and size of nodes represents number of genes. Edges represent GO-defined relations (similarity > 0.5), and thickness of edges represents similarity between gene sets. Interconnected synapse-related gene sets are circled and colored in orange. (D) GO terms enriched from downregulated proteins ( Usp7 cKO/WT fold change < 0.75). (E) Schematic of in vivo dendritic spine analysis of cortical pyramidal neurons. (F) Representative GFP micrographs of dendritic spines (arrowheads) on apical and basal dendrites. Scale bar, 5 μm. (G–I) Spine density on apical, basal, and all dendrites of pyramidal neurons in the motor cortex. **** p < 0.0001 by 2-tailed unpaired t test. Data are presented as mean ± SEM. See also .

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, an S protein-Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Quantitative Proteomics, In Vivo

Journal: Cell reports

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1016/j.celrep.2025.115231

Figure Lengend Snippet: (A) Domain structure of mouse USP7 protein. R105, D165, and W165 are three amino acids necessary for substrate binding. , USP, ubiquitin-specific protease domain; Ubl, ubiquitin-like domain. (B) Silver staining of the FLAG-immunoprecipitated USP7 TRAF domain (arrowhead) with and without RDW mutations (R105A+D165A+W166A). (C) Protein-protein association map of the top 50 USP7 TRAF interactors (full STRING network, confidence > 0.4). Proteins in complexes are colored according to (D). (D) GO analysis (cellular component) of the top 50 USP7 TRAF interactors. nBAF complex, neuronal SWI/SNF (BAF) complex. (E) Heatmap showing adjusted p values for enrichment of the USP7 TRAF interactors in disease genes. ID, intellectual disability; ADHD, attention deficit hyperactivity disorder; SCZ, schizophrenia; Ns, not significant. The p values were calculated by one-sided hypergeometric test with Benjamini-Hochberg correction. See also .

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, an S protein-Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Binding Assay, Ubiquitin Proteomics, Silver Staining, Immunoprecipitation

Journal: Cell reports

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1016/j.celrep.2025.115231

Figure Lengend Snippet: (A) Flowchart to characterize dynamics of the neuronal proteome in response to acute Usp7 knockout with 16-plex TMT-MS. (B) Immunoblot of USP7 depletion and Cre expression in primary cortical neurons over time. 14–3-3 is the loading control. (C) Volcano plot showing candidate substrates of USP7. Euclidean distances to USP7 were calculated in the space of TMT protein abundance. The p values were calculated by two-way ANOVA for Cre effect with Benjamini–Hochberg correction. (D) Heatmap of TMT protein abundance of USP7 and its candidate substrates. (E and F) Immunoblot images (E) and densitometric quantification (F) of candidate substrates in the cerebral cortex of Usp7 cKO; Trp53 +/− (n = 3) mice vs. Usp7 WT; Trp53 +/− ( n = 3) mice at P0. 14–3-3 and lamin A/C are loading controls. Arrowheads indicate multiple specific bands of a single candidate substrate. * p < 0.05, *** p < 0.001, **** p < 0.0001 by Šídák multiple-comparisons test. (G) RT-qPCR of candidate substrates in the cerebral cortex of Usp7 cKO; Trp53 +/− ( n = 5) mice vs. Usp7 WT; Trp53 +/− ( n = 3) mice at P0. **** p < 0.0001 by Šídák multiple-comparisons test. Data are presented as mean ± SEM. See also .

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, an S protein-Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Knock-Out, Western Blot, Expressing, Control, Quantitative Proteomics, Quantitative RT-PCR

Journal: Cell reports

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1016/j.celrep.2025.115231

Figure Lengend Snippet: (A) Representative GFP and PSD95 micrographs of dendritic spines (arrowheads) of DIV18 primary cortical neurons with knockdown of Ppil4 or luciferase (Luci). Micrographs of dendritic spines with knockdown of other candidate substrates are shown in . Scale bar, 5 μm. (B–G) Density of all (B–D) and PSD95+ (E–G) spines on apical, basal, and all dendrites of cortical neurons. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by Dunnett’s multiple-comparisons test (compared to Luci). (H) Streptavidin pull-down of HEK293T lysate with overexpression of streptavidin-binding peptide-FLAG (SFB)-tagged USP7 followed by immunoblot analyses. (I) IP of endogenous Ppil4 in HEK293T lysates followed by immunoblot analyses. (J) Reciprocal IP of endogenous USP7 and Ppil4 in mouse brain at age P4, followed by immunoblot analyses. Input lysate with long exposure for clear visualization is shown on the left. (K) TUBE pull-down of MG132-treated neuronal lysate with USP7 loss driven by lentiviral Cre followed by immunoblotting of Ppil4. Densitometric quantification is shown at the bottom. IB, immunoblot; a.u., arbitrary unit. (L and M) In vitro deubiquitination using FLAG-immunoprecipitated Ppil4 from MG132-treated HEK293T cells (L) and densitometric quantification (M) of high-molecular-weight Ppil4 over all Ppil4 signals with 3 biological replicates. ** p < 0.01 by 2-tailed unpaired t test. (N and O) Immunoblot and densitometric quantification ( n = 5 biological replicates) of endogenous Ppil4 in HEK293T cells expressing patient variants of USP7. USP7 C223A is catalytically dead as a positive control. * p < 0.05, ** p < 0.01 by Dunnett’s multiple-comparisons test after repeated-measurement one-way ANOVA. (P) Schematic of the USP7-Ppil4 signaling pathway regulating dendritic spines. Data are presented as mean ± SEM. See also .

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, an S protein-Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Knockdown, Luciferase, Over Expression, Binding Assay, Western Blot, In Vitro, Immunoprecipitation, High Molecular Weight, Expressing, Positive Control

Journal: Cell reports

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1016/j.celrep.2025.115231

Figure Lengend Snippet: (A) Flowchart to analyze RNA splicing upon USP7 loss. (B) GO terms enriched from genes of differential splicing clusters in LeafCutter analysis (FDR < 0.05 between Usp7 WT; Trp53 +/− and Usp7 cKO; Trp53 +/− mice). (C) Cumulative frequency curves of LeafCutter FDR of fully annotated and cryptic splicing clusters. Cumulative frequency curves for differential splicing clusters are expanded in the inset. * p < 0.05 by Kolmogorov-Smirnov test. (D) Percentage breakdown of exon-exon junction subtypes in differential and nondifferential splicing clusters. (E) Gene structure of Stxbp1 . (F–H) Inclusion of exon 19 of Stxbp1 , shown by LeafCutter sashimi plot (F), isoform-specific RT-qPCR bar plot (G), and volcano plot of all peptides mapped to Stxbp1 in TMT-MS (H). * p < 0.05 by uncorrected Fisher’s least significant difference (LSD) multiple-comparisons test. (I) Gene structure of Kalrn . (J–L) Usage of exon 34a or exon34b of Kalrn , shown by LeafCutter sashimi plot (J), isoform-specific RT-qPCR bar plot (K), and volcano plot of all peptides mapped to Stxbp1 in TMT-MS (L). **** p < 0.0001 by uncorrected Fisher’s LSD multiple-comparisons test. Data are presented as mean ± SEM. See also and .

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, an S protein-Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Quantitative RT-PCR

Journal: Cell reports

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1016/j.celrep.2025.115231

Figure Lengend Snippet:

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, an S protein-Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Western Blot, Immunoprecipitation, Immunofluorescence, Virus, Recombinant, Protease Inhibitor, Magnetic Beads, Mass Spectrometry, Software

Journal: Cell reports

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway.

doi: 10.1016/j.celrep.2025.115231

Figure Lengend Snippet: Figure 4. Interactome of the USP7 TRAF domain in cortical neurons (A) Domain structure of mouse USP7 protein. R105, D165, and W165 are three amino acids necessary for substrate binding.18,23 USP, ubiquitin-specific protease domain; Ubl, ubiquitin-like domain. (B) Silver staining of the FLAG-immunoprecipitated USP7 TRAF domain (arrowhead) with and without RDW mutations (R105A+D165A+W166A). (C) Protein-protein association map of the top 50 USP7 TRAF interactors (full STRING network, confidence > 0.4). Proteins in complexes are colored according to (D). (D) GO analysis (cellular component) of the top 50 USP7 TRAF interactors. nBAF complex, neuronal SWI/SNF (BAF) complex. (E) Heatmap showing adjusted p values for enrichment of the USP7 TRAF interactors in disease genes. ID, intellectual disability; ADHD, attention deficit hyperactivity disorder; SCZ, schizophrenia; Ns, not significant. The p values were calculated by one-sided hypergeometric test with Benjamini-Hochberg correction. See also Figure S5.

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, an S protein-Streptavidin-binding peptideFLAG (SFB)-taggedUSP7 construct61 (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Binding Assay, Ubiquitin Proteomics, Silver Staining, Immunoprecipitation

Journal: Cell reports

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway.

doi: 10.1016/j.celrep.2025.115231

Figure Lengend Snippet: Figure 5. Acute USP7 loss transforms the proteome of cortical neurons and depletes candidate substrate at the protein level (A) Flowchart to characterize dynamics of the neuronal proteome in response to acute Usp7 knockout with 16-plex TMT-MS. (B) Immunoblot of USP7 depletion and Cre expression in primary cortical neurons over time. 14-3-3 is the loading control.

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, an S protein-Streptavidin-binding peptideFLAG (SFB)-taggedUSP7 construct61 (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Knock-Out, Western Blot, Expressing, Control

Journal: Cell reports

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway.

doi: 10.1016/j.celrep.2025.115231

Figure Lengend Snippet: Figure 6. USP7 targets and deubiquitinates Ppil4 to promote dendritic spinogenesis (A) Representative GFP and PSD95 micrographs of dendritic spines (arrowheads) of DIV18 primary cortical neurons with knockdown of Ppil4 or luciferase (Luci). Micrographs of dendritic spines with knockdown of other candidate substrates are shown in Figure S8. Scale bar, 5 mm. (B–G) Density of all (B–D) and PSD95+ (E–G) spines on apical, basal, and all dendrites of cortical neurons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Dunnett’s multiple-comparisons test (compared to Luci). (H) Streptavidin pull-down of HEK293T lysate with overexpression of streptavidin-binding peptide-FLAG (SFB)-tagged USP7 followed by immunoblot analyses. (I) IP of endogenous Ppil4 in HEK293T lysates followed by immunoblot analyses. (J) Reciprocal IP of endogenous USP7 and Ppil4 in mouse brain at age P4, followed by immunoblot analyses. Input lysate with long exposure for clear visualization is shown on the left. (K) TUBE pull-down of MG132-treated neuronal lysate with USP7 loss driven by lentiviral Cre followed by immunoblotting of Ppil4. Densitometric quantification is shown at the bottom. IB, immunoblot; a.u., arbitrary unit.

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, an S protein-Streptavidin-binding peptideFLAG (SFB)-taggedUSP7 construct61 (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Knockdown, Luciferase, Over Expression, Binding Assay, Western Blot

Journal: bioRxiv

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1101/2023.10.24.563880

Figure Lengend Snippet: Loss of USP7 in glutamatergic excitatory neurons leads to multiple disease-relevant behavioral deficits and apoptosis in mice. (A) Patients with the Hao-Fountain syndrome and Usp7 conditional knockout (cKO) mice exhibit phenotypes in similar behavioral domains. (B) Double immunofluorescence of USP7 and neuron nuclear protein (NeuN) in cerebral cortex at postnatal day 42 (P42). USP7 protein was absent in most neuronal nuclei in Usp7 cKO cortex. Scale bar 100µm. (C) Virtual optomotor system (VOS) test: Spatial frequency threshold for head turn. Usp7 cKO mice (n = 6) showed reduced visual acuity compared to Usp7 WT (n = 5) and cHet mice (n = 8). **p<0.01 by Bonferroni’s multiple comparison test. (D) Motor strength test: Time to grip onto inverted screen. Usp7 cKO mice (n = 8) showed shorter latency to fall than Usp7 WT (n = 8) and cHet mice (n = 11). *p<0.05, **p<0.01 by Dunn’s multiple comparison test. (E) Catwalk 1: Relative mean intensity distributed onto each paw. Usp7 cKO mice (n = 8) put less weight onto front paw and more onto hind paw, compared to Usp7 WT (n = 7) and cHet mice (n = 9). **p<0.01 by Tukey’s multiple comparison test. See also Video S2. (F) Catwalk 2: Limb swing speed. Usp7 cKO mice (n = 8) showed higher swing speed of both front and hind limb than Usp7 WT (n = 7) and cHet mice (n = 9). **p<0.01, ***p<0.001 by Tukey’s multiple comparison test. See also Video S2. (G) Fear conditioning day 1: Time spent in freezing for tone/shock pairing (n = 8, 11 & 6 for Usp7 WT, Usp7 cHet and Usp7 cKO mice). No significant genotype effect by two-way ANOVA. (H) Fear conditioning day 2: Time spent in freezing for contextual conditioning. Usp7 cKO mice spent less time in freezing than Usp7 WT mice. Numbers of mice are the same as in (G). *p<0.05, **p<0.01 by Bonferroni’s multiple comparison test ( Usp7 WT vs. cKO). (I) Fear conditioning day 3: Time spent in freezing for auditory cue conditioning. Usp7 cKO mice spent less time in freezing after the tone than Usp7 WT mice. Numbers of mice are the same as in (G). *p<0.05 by Bonferroni’s multiple comparison test ( Usp7 WT vs. cKO). (J) Percentage of tail biters among all monitored Usp7 cKO mice from 3 to 12 weeks old. Tail biters were identified if their co-caged mice had biting marks on the tails. (K) Representative biting injury on mice co-caged with Usp7 cKO mice. See also Video S3. Data are presented as mean ± SEM. See also .

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, a Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Knock-Out, Immunofluorescence, Comparison

Journal: bioRxiv

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1101/2023.10.24.563880

Figure Lengend Snippet: Additional phenotypic characterization of Usp7 cKO mice. Related to and . (A) Immunofluorescence of USP7 in cerebral cortex at embryonic day 14 (E14). USP7 is localized to both Ki-67+ ventricular-subventricular zone (V-SVZ) and cortical plate (CP) in USP7 WT cortex. But in Usp7 cKO cortex, USP7 is depleted only in post-mitotic cortical plate (dashed outline). Scale bar 100µm. (B) Body weight curve from P6 to P48. Usp7 cKO mice (n = 7) exhibited body weight growth delay, compared to Usp7 WT (n = 10) and cHet mice (n = 7). (C) Body weight of adult mice. Number of mice = 8, 11 & 9 for Usp7 WT, cHet & cKO, respectively. No significant genotype effect by one-way ANOVA. (D) Motor coordination test 1: Ledge test. Usp7 cKO mice (n = 8) showed shorter latency to fall from ledge than Usp7 WT (n = 7) and cHet mice (n = 9). **p<0.01 by Dunn’s multiple comparison test. (E) Motor coordination test 2: Platform test. Usp7 cKO mice showed shorter latency to fall from platform than Usp7 WT mice. Numbers of mice are the same as in (D). **p<0.01 by Dunn’s multiple comparison test. (F) Motor coordination test 3: Pole test, time to climb down the pole. Numbers of mice are the same as in (D). No significant genotype effect by one-way ANOVA. (G) Motor coordination test 4: Pole test, time to turn at the tip of the pole. Usp7 cKO mice spent more time in turning than Usp7 WT and cHet mice. Numbers of mice are the same as in (D). *p<0.05, **p<0.01 by Dunn’s multiple comparison test. (H) Catwalk 3: Stride length. Usp7 cKO mice showed slightly shorter average stride length than control. Numbers of mice are the same as in (D). *p<0.05 by Tukey’s multiple comparison test. (I) Catwalk 4: Swing time. Usp7 cKO mice used less time swinging front and hind legs for each step, compared to Usp7 WT and cHet mice. Numbers of mice are the same as in (D). *p<0.05, ***p<0.001, ****p<0.0001 by Tukey’s multiple comparison test. (J) Hearing test: Magnitude of response to acoustic startle at different dB level. Number of mice = 5, 8 & 6 for Usp7 WT, Usp7 cHet & Usp7 cKO. No significant genotype effect by two-way ANOVA. (K) Y maze: Alternative rate in choosing arm to enter. Number of mice = 4, 6 & 5 for Usp7 WT, Usp7 cHet & Usp7 cKO. No significant genotype effect by one-way ANOVA. Data are presented as mean ± SEM.

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, a Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Immunofluorescence, Comparison

Journal: bioRxiv

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1101/2023.10.24.563880

Figure Lengend Snippet: Deletion of p53 rescues apoptosis, impairment of visual acuity and auditory cue conditioning in Usp7 cKO mice but not other behavioral deficits. (A) Immunofluorescence of apoptosis marker cleaved caspase 3 (c-cas 3) in cerebral cortex at different developmental time points. Usp7 cKO cortex showed large scale of apoptosis at P0, whereas apoptosis is low at E15, P5 and P10. Scale bars all 200µm. (B) Schematic of USP7-Mdm2-p53 signaling pathway regulating apoptosis. (C) Immunofluorescence of c-cas 3 in P0 cortex of different Trp53 and Usp7 genotypes. Trp53 heterozygosity diminished USP7 loss-induced apoptosis. Scale bar 200µm. (D) Quantification of c-cas 3 fluorescent signal, as shown in (C). Number of mice = 3, 3, 4 & 3 for Usp7 WT, Usp7 cKO, Usp7 cKO; Trp53 +/− & Usp7 WT; Trp53 +/−, respectively. *p<0.05 by Tukey’s multiple comparison test. (E) Virtual optomotor system (VOS) test: Spatial frequency threshold for head turn. Usp7 cKO; Trp53 +/− mice (n = 15) did not have impaired visual acuity, compared to Usp7 WT; Trp53 +/− (n = 14) and Usp7 cHet; Trp53 +/− mice (n = 9). No significant genotype effect by one-way ANOVA. (F) Motor strength test: Time to grip onto inverted screen. Usp7 cKO mice (n = 10) showed shorter latency to fall than Usp7 WT; Trp53 +/− (n = 10) and Usp7 cHet; Trp53 +/− mice (n = 14). *p<0.05, **p<0.01 by Dunn’s multiple comparison test. (G) Catwalk 1: Relative mean intensity distributed onto each paw. Usp7 cKO; Trp53 +/− mice (n = 5) put less weight onto front paw and more onto hind paw than Usp7 WT; Trp53 +/− (n = 7) and Usp7 cHet; Trp53 +/− mice (n = 8). ***p<0.001, ****p<0.0001 by Tukey’s multiple comparison test. See also Video S4. (H) Catwalk 2: Limb swing speed. Usp7 cKO; Trp53 +/− mice showed slightly higher swing speed than Usp7 cHet; Trp53 +/− mice. Numbers of mice are the same as in (G). *p<0.05 by Tukey’s multiple comparison test. See also Video S4. (I) Fear conditioning day 1: Time spent in freezing for tone/shock pairing (n = 12, 6 & 11 for Usp7 WT, Usp7 cHet and Usp7 cKO mice). No significant genotype effect by two-way ANOVA. (J) Fear conditioning day 2: Time spent in freezing for contextual conditioning. Usp7 cKO; Trp53 +/− mice spent less time in freezing than Usp7 WT; Trp53 +/− mice. Numbers of mice are the same as in (H). *p<0.05, by Bonferroni’s multiple comparison test ( Usp7 WT vs. cKO). (K) Fear conditioning day 3: Time spent in freezing for auditory cue conditioning. Usp7 cKO; Trp53 +/− mice had no difference in freezing after the tone, compared to Usp7 WT; Trp53 +/− and Usp7 cHet; Trp53 +/− mice. Numbers of mice are the same as in (H). No significant genotype effect by two-way ANOVA. (L) Percentage of tail biters among all monitored Usp7 cKO; Trp53 +/− mice from 3 to 12 weeks old. Tail biters were counted if their co-caged mice had biting marks on their tails. (M) Representative biting injury on mice co-caged with Usp7 cKO; Trp53 +/− mice. Data are presented as mean ± SEM. See also , and .

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, a Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Immunofluorescence, Marker, Comparison

Journal: bioRxiv

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1101/2023.10.24.563880

Figure Lengend Snippet: Loss of USP7 in glutamatergic neurons induces apoptosis in a cell autonomous manner. Related to . (A) Double immunofluorescence of cleaved caspase 3 (c-cas 3) and Cre. An expanded region shows overlap (arrows) of cleaved caspase 3 and Cre, supporting that the apoptotic cells are glutamatergic neurons. Scale bar 200µm. (B) Western blot validation of USP7 RNAi in HEK293Tcells. Both USP7i.1 and USP7i.2 diminished expression of GFP-USP7. 14-3-3 is loading control. (C) Immunocytochemistry of c-cas 3 in primary cortical neurons dissociated from wild-type E14 cortices and transfected with a GFP plasmid. Scale bar 100µm. Cells were fixed at DIV5, 6 and 7. At the bottom are close-up of neurons with and without c-cas 3 signal. (D) Quantification of apoptotic percentage, defined as the percentage of c-cas 3 and GFP double positive neurons among all GFP positive neurons. Cortical neurons were transfected with USP7 shRNA plasmids at 3 days in vitro (DIV3) and analyzed at DIV5, 6 and 7. Data were collected from three independent batches of neuron culture, three wells per condition within each batch. USP7 knockdown by two independent shRNA led to a progressive elevation in apoptotic percentage. Empty vector U6 and knockdown of an irrelevant deubiquitinase, USP30, served as negative controls. ***p<0.001, ****p<0.0001 by Tukey’s multiple comparison test. Data are presented as mean ± SEM.

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, a Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Immunofluorescence, Western Blot, Expressing, Immunocytochemistry, Transfection, Plasmid Preparation, shRNA, In Vitro, Comparison

Journal: bioRxiv

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1101/2023.10.24.563880

Figure Lengend Snippet: Deletion of p53 rescues USP7 loss-induced apoptosis but minimally restores the brain volume reduction in Usp7 cKO mice. Related to and . (A) Immunofluorescence of c-cas 3 in the cerebral cortex of Usp7 cKO mice at P0 with deletion of one or both allele of Trp53 . C-cas 3 signal remained sparse with either Trp53 heterozygosity or homozygosity. Scale bar 200µm. (B) Immunofluorescence of USP7 in primary cortical neurons dissociated from Usp7 fl/fl ; Trp53 +/− embryos. Neurons transduced with Cre WT showed loss of USP7 protein at DIV14. Scale bar 25µm. (C) MTS assay of Usp7 fl/fl cortical neurons transduced by lentiviral Cre with different Trp53 genotypes. USP7 knockout in Trp53 +/+ neurons (n = 5 wells) strongly reduced cell viability, indicating of neuronal death. No significant viability decrease in Trp53 +/− (n = 3 wells) and Trp53 −/− neurons (n = 5 wells). Puromycin (2µg/mL, 24hrs) treated neurons serve as positive control. ***p<0.001 by Šídák’s multiple comparisons test between Cre WT and Cre dead. (D) Representative segmentation on FA map and DWI for volumetric measurement with diffusion-weighted ex vivo MRI. Cerbral cortex and hippocampus are shown in blue and red, respectively. (E) Volumetric measurement with ex vivo MRI. Usp7 cKO brains (P30-32) are smaller than Usp7 WT . Deletion of p53 minimally stored the volume of cortex and hippocampus. OB = olfactory bulbs. Number of mice = 5, 4, 5 & 5 for Usp7 WT, Usp7 cKO, Usp7 WT; Trp53 +/− & Usp7 cKO; Trp53 +/− . *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Tukey’s multiple comparison test. Data are presented as mean ± SEM.

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, a Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Immunofluorescence, Transduction, MTS Assay, Knock-Out, Positive Control, Diffusion-based Assay, Ex Vivo, Comparison

Journal: bioRxiv

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1101/2023.10.24.563880

Figure Lengend Snippet:

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, a Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Comparison

Journal: bioRxiv

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1101/2023.10.24.563880

Figure Lengend Snippet: Additional phenotypic characterization of Usp7 cKO; Trp53 +/− mice. Related to and . (A) Body weight curve from P6 to P48. Usp7 cKO; Trp53 +/− mice (n = 6) exhibited body weight growth delay, compared to Usp7 WT; Trp53 +/− (n = 10) and Usp7 cHet; Trp53 +/− mice (n = 5). (B) Body weight of adult mice. Number of mice = 10, 14 & 10 for Usp7 WT, cHet & cKO; Trp53 +/− , respectively. No significant genotype effect by one-way ANOVA. (C) Motor coordination test 1: Ledge test. Usp7 cKO; Trp53 +/− mice showed slightly shorter latency to fall from ledge than control mice. Numbers of mice are the same as in (B). *p<0.05 by Dunn’s multiple comparison test. (D) Motor coordination test 2: Platform test. Usp7 cKO; Trp53 +/− mice showed shorter latency to fall from platform than control mice. Numbers of mice are the same as in (B). **p<0.01 by Dunn’s multiple comparison test. (E) Motor coordination test 3: Pole test, time to climb down the pole. Numbers of mice are the same as in (B). No significant genotype effect by one-way ANOVA. (F) Motor coordination test 4: Pole test, time to turn at the tip of the pole. Usp7 cKO; Trp53 +/− mice spent more time in turning than control mice. Numbers of mice are the same as in (B). **p<0.01, ***p<0.001 by Dunn’s multiple comparison test. (G) Catwalk 3: Stride length. Usp7 cKO; Trp53 +/− mice showed shorter average stride length than control. Number of mice = 7, 8 & 5 for Usp7 WT, cHet & cKO; Trp53 +/− . *p<0.05, **p<0.01 by Tukey’s multiple comparison test. (H) Catwalk 4: Swing time. Usp7 cKO; Trp53 +/− mice used less time swinging front and hind legs for each step, compared to control mice. Numbers of mice are the same as in (G). *p<0.05, ***p<0.001 by Tukey’s multiple comparison test. (I) Hearing test: Magnitude of response to acoustic startle at different dB level. Number of mice = 10, 14 & 10 for Usp7 WT, cHet & cKO; Trp53 +/− . No significant genotype effect by two-way ANOVA. Data are presented as mean ± SEM.

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, a Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Comparison

Journal: bioRxiv

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1101/2023.10.24.563880

Figure Lengend Snippet: Loss of USP7 reduces synapses in cortex. (A) Flowchart to profile the proteome of the cerebral cortex of Usp7 cKO mice with tandem mass tag-mass spectrometry (TMT-MS). (B) Volcano plot of all profiled proteins in TMT-MS. Up-regulated and down-regulated proteins ( Usp7 cKO vs. WT) are labeled in red and blue, respectively. P-value were calculated by 2-tail unpaired t-test. (C) Organic layout of enriched GO gene sets based on TMT protein abundance in (B). Nodes represent gene sets (q-value < 0.01) and size of nodes represents how many genes contained in gene sets. Edges represent GO-defined relations (similarity > 0.5) and thickness of edges represents similarity between gene sets. Most enriched gene sets are related to synapse and circled in red. All gene sets shown are enriched in Usp7 WT and de-enriched in Usp7 cKO. (D) GO terms enriched from ranked list of down-regulated proteins ( Usp7 cKO vs. WT fold change < 0.75). (E) Ventricular injection of AAV PHP.eB Syn-GFP-P2A-GFPf at P0/1 to sparsely label pyramidal neurons in cerebral cortex and dendritic spine analysis at P18. (F) Representative images of dendritic spines on apical and basal dendrites. Scale bar 5µm. (G, H, I) Spine density on apical, basal and all dendrites of pyramidal neurons in motor cortex. Usp7 cKO; Trp53 +/− neurons (n = 39 & 39 neurons for apical dendrites & basal dendrites from 3 mice) showed reduced spine density than Usp7 WT; Trp53 +/− neurons (n = 40 & 50 neurons for apical dendrites & basal dendrites from 3 mice). ****p<0.0001 by 2-tail unpaired t-test. Data are presented as mean ± SEM. See also .

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, a Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Mass Spectrometry, Labeling, Injection

Journal: bioRxiv

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1101/2023.10.24.563880

Figure Lengend Snippet: USP7 loss does not cause significant change in dendritic morphology in primary cortical neurons with Trp53 heterzygosity. Related to . (A) Representative DIV11 images of cortical pyramidal neurons and their Imaris dendritic traces. For the Imaris dendritic traces, apical dendrties are highlighted in yellow. The cortical neurons were dissociated from Usp7 fl/fl ; Trp53 +/− E15 embryos and transduced with Cre WT or Cre dead lentivirus at DIV3. (B) Sholl profile of pyramidal neurons (n = 40 & 32 for Cre dead & WT) and all neurons (n = 55 & 44 for Cre dead & WT). Shaded area represents standard deviation. Profiles of neurons transduced with Cre dead and Cre WT are colored in red and green, respectively. (C) Sum of dendrite length in pyramidal neurons, apical dendrites vs. basal dendrites (n = 40 & 32 for Cre dead & WT). No significant Cre effect by two-way ANOVA. (D) Sum of dendrite length in pyramidal neurons, across 1-4 branch levels. Numbers of neurons are the same as in (C). No significant Cre effect by two-way ANOVA. (E) Average length of primary dendrites in pyramidal neurons. Numbers of neurons are the same as in (C). No significance by 2-tail unpaired t-test. (F) Number of primary dendrites in pyramidal neurons. Numbers of neurons are the same as in (C). No significance by 2-tail unpaired t-test. (G) Soma 3D-volumes of pyramidal neurons (n = 65 & 57 for Cre dead & WT). No significance by 2-tail unpaired t-test. Data are presented as mean ± SEM.

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, a Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Transduction, Standard Deviation

Journal: bioRxiv

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1101/2023.10.24.563880

Figure Lengend Snippet: Identification of proteins that interact with the USP7-TRAF domain and are stabilized by USP7. (A) Domain structure of mouse USP7 protein. R105, D165 and W165 on the TRAF domain are three amino acids critical for substrate binding , . USP: Ubiquitin specific protease domain. Ubl: Ubiquitin like domain. (B) Silver stain of FLAG-immunoprecipitated USP7-TRAF domain (arrow) with and without RDW mutations (R105A + D165A + W166A).1.5% of all eluted sample were stained while rest of the sample were analyzed by liquid chromatography-mass spectrometry (LC-MS). (C) Protein-protein association map of USP7-TRAF interactors (full STRING network, confidence > 0.4). Edge thickness represents confidence in association between proteins. Shown are only 50 interactors with highest gene length-normalized spectral counts. Ribonucleoprotein complex is colored in red. Spliceosomal complex is colored in green. SWI/SNF complex is colored in blue. Mitochondrial pyruvate dehydrogenase complex is colored in yellow. (D) Molecular component GO enrichments of top 50 interactors of the USP7-TRAF domain. Different molecular components are colored as in (C). (E) Flowchart to profile proteomic change with tandem mass tag-mass spectrometry (TMT-MS) in response to acute Usp7 knockout in primary cortical neurons. Neurons were dissociated from Usp7 fl/fl ; Trp53 −/− embryos at E15 and subject to Cre lentiviral transduction at DIV3. Cre harboring Y331F mutation is catalytically dead (Cre dead) and served as negative control. (F) Western blot validation of USP7 depletion and Cre expression in Cre WT-transduced neurons over time. 14-3-3 is loading control. (G) Heatmap of TMT protein abundance of USP7 and its candidate substrates. Candidate substrates were sorted out by smallest p-value of Cre main effect (two-way ANOVA) and shortest Euclidean distance to USP7. (H) Western blot quantitation of candidate substrates with the cerebral cortex of Usp7 cKO; Trp53 +/− (n = 3 mice) vs. Usp7 WT; Trp53 +/− (n = 3 mice) at P0. Western blot images are in . (I) RT-qPCR of candidate substrates with the cerebral cortex of Usp7 cKO; Trp53 +/− (n = 5 mice) vs. Usp7 WT; Trp53 +/− (n = 3 mice) at P0. No candidate substrate in the analysis showed RNA reduction with the loss of USP7. Data are presented as mean ± SEM. See also , and .

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, a Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Binding Assay, Silver Staining, Immunoprecipitation, Staining, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Knock-Out, Transduction, Mutagenesis, Negative Control, Western Blot, Expressing, Quantitation Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1101/2023.10.24.563880

Figure Lengend Snippet: Validation of USP7 TRAF domain expression and interaction for IP-MS. Related to . (A) Immunoprecipitation of GFP-TRAF domain followed by western blot of myc3-Mdm2 and GFP. Lysates were prepared from HEK293T cells transfected with myc3-Mdm2 and GFP-tagged USP7-TRAF domain. DW double and RDW triple mutations (All mutated into Alanine) on the USP7-TRAF domain disrupted the interaction with MDM2. FT as flow-through lysates. (B) Mostly nuclear localization of truncated GFP-USP7 in rat cortical neurons at DIV9, including USP7-TRAF domain (USP7 amino acid 55-209). Neurons were co-transfected with pCAG-mCherry with Ca phosphate. USP7 fl as USP7 full length. Scale bar 50µm. (C) Immunocytochemistry of FLAG showed mostly nuclear expression of FLAG-USP7 TRAF domain in rat cortical neurons with and without RDW mutations at DIV10. Immunocytochemistry of Map2 shows neuronal morphology. Scale bar 100µm.

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, a Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Expressing, Immunoprecipitation, Western Blot, Transfection, Immunocytochemistry

Journal: bioRxiv

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1101/2023.10.24.563880

Figure Lengend Snippet: A closer look at the in vitro TMT-MS results. Related to . (A) PCA of 16 samples based on TMT abundance of all 8304 proteins detected by TMT-MS. Principal component 1 (PC1) separates DIV6 and DIV8 samples whereas principal component 2 (PC2) separates Cre WT and Cre dead samples. (B) TMT protein abundance of candidate substrates in previous in vivo TMT-MS . Most candidate substrates showed various degrees of reduction in the cerebral cortex of Usp7 cKO mice at P17. n = 5 for both Usp7 WT; Trp53 +/− and Usp7 cKO; Trp53 +/− mice. (C) Dendrogram and heatmap of TMT protein adjacencies by weighted gene co-expression network analysis (WGCNA). WGCNA segragates 2627 differentially expressed proteins (Cre effect p<0.05 or Cre time interaction effect p<0.05 by two-way ANOVA) into 10 modules in different colors. (D) Module-trait relationships. Correlations and p-values between module eigengene (ME) and Cre (Cre WT as 1, Cre dead as 0) or time (DIV6 or DIV8) are denoted and color-coded according to the color legend. (E) Membership of candidate substrates in each module. (F) GO annotation for each module. Turquiose module is enriched in ribonucleoprotein complex and splicing-related terms. Brown module is enriched in microtubule and axon-related terms. Black module is enriched in mitochondrion-related terms. Yellow module is enriched in E3 ligase-related terms. Data are presented as mean ± SEM.

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, a Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: In Vitro, In Vivo, Expressing

Journal: bioRxiv

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1101/2023.10.24.563880

Figure Lengend Snippet: Western blot with P0 Usp7 cKO; Trp53 +/− cortex. Related to . Three biological replicates of each genotype were denoted in numbers. (A) Phip, USP7 and Xiap. (B) Ppil4, Ring1 and Gpd1. (C) Ubxn7, Ruvbl2 and Rnf2. (D) Chfr and Sub1. (E) Fto, Gfap and Cre. (F) Rnf220, Cfdp1 and Phc. (G) Fam172a. (H) Trip12. (I) Smarcal1. 14-3-3 and Lamin A/C are loading controls.

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, a Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Western Blot

Journal: bioRxiv

Article Title: The Hao-Fountain syndrome protein USP7 regulates neuronal connectivity in the brain via a novel p53-independent ubiquitin signaling pathway

doi: 10.1101/2023.10.24.563880

Figure Lengend Snippet: USP7 targets and de-ubiquitinates Ppil4 to promote synaptogenesis. (A) Streptavidin pull-down of HEK293T lysate with overexpression of Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 followed by western blot of multiple endogenous candidate substrates. 2% input lysate is shown on the left. (B, C, D) Density of dendritic spine on apical, basal and all dendrites of pyramidal-shaped neurons in (H). Two independent lentiviral shRNA knocking down Ppil4 reduced spine density compared with control shRNA targeting Luciferase (Luci). Number of neurons (apical dendrties) = 24, 27, 23, 20, 23, 18, 16 & 17 for Luci, Ppil4i.1, Ppil4i.2, Smarcal1i.1, Smarcal1i.2, Trip12i, Ring1i & Sub1i. Number of neurons (basal dendrties) = 22, 24, 26, 20, 24, 17, 15 & 16 for Luci, Ppil4i.1, Ppil4i.2, Smarcal1i.1, Smarcal1i.2, Trip12i, Ring1i & Sub1i. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Dunnett’s multiple comparison test (compared to Luci). (E, F, G) Density of PSD95+ dendritic spine on apical, basal and all dendrites of cortical neurons in (H) and . Two independent lentiviral shRNA knocking down Ppil4 reduced PSD95+ spine density compared with control shRNA targeting Luciferase (Luci). Number of neurons are the same as in (B, C, D). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Dunnett’s multiple comparison test (compared to Luci). (H) Representative images show the effect of Ppil4 knockdown on dendritic spines of cortical neurons at DIV18. PSD95 puncta decorating the dendritic spines were immunostained along with GFP. Arrows show dendritic spines. Scale bar 5µm. (I) Immunoprecipitation of endogenous Ppil4 in HEK293T lysates followed by western blot of GFP-USP7 and Ppil4. 2% Input lysate is shown on the left. GFP-USP7 was co-immunoprecipiated with Ppil4. (J) Tandem Ubiquitin Binding Entities (TUBE) pull-down of neuronal lysate with USP7 loss driven by lentiviral Cre, followed by immunoblotting of Ppil4. Input lysates are shown at the bottom. Endogenous Poly-Ubiquitinated Ppil4 increased with USP7 loss (Cre WT). A.U. = aribitary unit. (K) In vitro deubiquitination assay using FLAG-immunoprecipitated Ppil4 from HEK293T cells. Incubation with recombinant His-USP7 at 37°C for 1 hour converted high-molecular weight ubiquitinated Ppil4 (dashed rectangle) into low-molecular weight Ppil4 (arrow). (L) Densitometric quantification of high-molecular weight Ppil4 over all Ppil4 signal in (K) over 3 biological replicates. **p<0.01 by 2-tail unpaired t-test. (M) Western blot of USP7 with mutations from the Hao-Fountain syndrome patients in HEK293T cells. USP7 C223A is catalytically dead. (N) Western blot of endogenous Ppil4 in HEK293T cells expressing different USP7 variants. (O) Densitometric quantification of Ppil4 signal as in (N) over 4 biological replicates. Repeated measurement one way ANOVA. *p<0.05 by Dunnett’s multiple comparison. (P) Schematic of USP7-Ppil4 signaling pathway regulating dendritic spine density in glutamatergic cortical neuron. Data are presented as mean ± SEM. See also .

Article Snippet: For assessment of the interaction between USP7 and endogenously expressed proteins, a Streptavidin-binding peptide-FLAG (SFB)-tagged USP7 construct (Addgene plasmid #99393) was transfected into HEK293T cells via PEI transfection.

Techniques: Over Expression, Binding Assay, Western Blot, shRNA, Luciferase, Comparison, Immunoprecipitation, In Vitro, Incubation, Recombinant, Molecular Weight, Expressing