Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: E6AP downregulates p53 levels in an HCV Core-dependent manner. ( a – d ) HepG2 and Hep3B cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the indicated plasmids, for 48 h, followed by Western blotting. The images of the target proteins were cropped from the original images and band intensities were quantified using ImageJ (NIH). Values indicate p53 and E6AP levels relative to the loading control (γ-tubulin).

Article Snippet: Scrambled (SC) small hairpin RNA (shRNA; #sc-42964) and E6AP shRNA (#sc-43742) plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Control

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: HCV Core stimulates E6AP-mediated proteasomal degradation of p53 but inhibits MDM2-mediated degradation. HepG2 and Hep3B cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the indicated plasmids, for 48 h. For ( a ), HepG2-Core cells stably expressing HCV Core were also included. ( a , b ) Protein levels were measured by Western blot analysis. ( c ) Cells were treated with 50 μM cycloheximide (CHX) for the indicated times before harvesting, followed by Western blotting. Bands of p53 and γ-tubulin were quantified to determine the half-life (t1/2) of p53. The difference in the p53-to-γ-tubulin ratio among samples is shown in a graph. ( d , e ) Cells were treated with 10 μM MG132 for 4 h before harvesting to block further proteasomal degradation. For lanes 5 and 6 in ( e ), cells were treated with Nutlin 3a for 24 h. Total p53 was immunoprecipitated with an anti-p53 antibody and subjected to Western blotting using anti-E6AP, anti-MDM2, anti-HCV Core, and anti-HA antibodies to detect E6AP, MDM2, HCV Core, and HA-Ub-complexed p53, respectively. The input indicates the levels of the designated proteins in the cell lysates. ( f , g ) For mammalian two-hybrid assays, Hep3B cells were transfected with the Gal4 reporter (G5E1b-luc), pSG424-E6AP (or pSG424-MDM2), and pCMV p53-VP16, along with the indicated plasmids, for 48 h, followed by a luciferase assay. For ( f ), cells were treated with 10 μM MG132 for 4 h before harvesting. Luciferase activity from G5E1b-luc was normalized to the β-gal activity measured in the corresponding cell extract. The values show relative luciferase activity compared to the control’s basal level. Results are presented as mean ± SD from four independent experiments ( n = 4). ND, not detected.

Article Snippet: Scrambled (SC) small hairpin RNA (shRNA; #sc-42964) and E6AP shRNA (#sc-43742) plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Stable Transfection, Western Blot, Blocking Assay, Immunoprecipitation, Luciferase, Activity Assay

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: E6AP induces ubiquitin-dependent proteasomal degradation of phosphorylated p53 in the presence of HCV Core. HepG2 and Hep3B cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the designated plasmids, for 48 h. For ( c , d ), cells were treated with 10 μM MG132 for 4 h before harvesting. Band intensities were quantified using ImageJ (NIH). ( a , d ) Protein levels were measured by Western blot analysis. ( b ) Phosphorylated p53 at Ser-15 (pSer-15 p53) in cell lysates was immunoprecipitated with an anti-pSer-15 p53 antibody, followed by Western blotting to detect E6AP, MDM2, HCV Core, and HA-Ub-complexed pSer-15 p53. The input shows the levels of the indicated proteins in cell lysates. ( c ) E6AP or MDM2 were immunoprecipitated with the appropriate antibodies and subjected to Western blotting to detect HCV Core, p53, pSer-15 p53, and pSer-20 p53. The input shows the levels of the indicated proteins in cell lysates. ND, not detected.

Article Snippet: Scrambled (SC) small hairpin RNA (shRNA; #sc-42964) and E6AP shRNA (#sc-43742) plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: HCV Core-induced p53 phosphorylation is required for E6AP-mediated proteasomal degradation. ( a ) HepG2 cells transiently transfected with the designated plasmids for 47 h were treated with the indicated concentrations of the ATM inhibitor KU-55933 for 1 h before harvesting, followed by Western blotting. ( b , c ) Cell lysates prepared in ( a ) were immunoprecipitated with anti-p53 or anti-E6AP antibodies, followed by Western blotting. ( d – f ) For mammalian two-hybrid assays, Hep3B cells were transfected with pSG424-E6AP (or pSG424-MDM2), pCMV p53-VP16, and G5E1b-luc, along with the indicated plasmids, for 47 h. Cells were either mock-treated or treated with 10 μM KU-55933 for 1 h before harvesting, followed by a luciferase assay ( n = 4). ND, not detected.

Article Snippet: Scrambled (SC) small hairpin RNA (shRNA; #sc-42964) and E6AP shRNA (#sc-43742) plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Transfection, Western Blot, Immunoprecipitation, Luciferase

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: p53 phosphorylation is sufficient for E6AP to induce p53 ubiquitination. ( a ) HepG2 cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the designated amounts of the E6AP expression plasmid, for 48 h. Cells were treated with the designated concentrations of etoposide for 24 h and MG132 for 4 h before harvesting, followed by Western blotting. ( b – d ) Cell lysates prepared in ( a ) were immunoprecipitated with anti-p53, anti-pSer-15 p53, or anti-E6AP antibodies, followed by Western blotting. ( e – g ) Hep3B cells were transfected with pSG424-E6AP (or pSG424-MDM2), pCMV p53-VP16, and G5E1b-luc, along with the indicated plasmids, for 48 h. Cells were either mock-treated or treated with 10 μM etoposide for 24 h before harvesting, followed by a luciferase assay ( n = 4).

Article Snippet: Scrambled (SC) small hairpin RNA (shRNA; #sc-42964) and E6AP shRNA (#sc-43742) plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Luciferase

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: Phosphorylation of p53 at Ser-15 is crucial for E6AP-mediated protein degradation. Hep3B cells were transfected with either wild-type (WT) p53 or p53 mutants with substitutions at Ser-15 and/or Ser-20, along with the indicated plasmids, for 48 h. ( a , b , d ) Protein levels were determined by Western blotting. ( c , e ) Cell lysates were immunoprecipitated with an anti-p53 antibody, and the immunoprecipitates were analyzed by Western blotting.

Article Snippet: Scrambled (SC) small hairpin RNA (shRNA; #sc-42964) and E6AP shRNA (#sc-43742) plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Transfection, Western Blot, Immunoprecipitation

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: HCV Core triggers E6AP-dependent ubiquitination of p53 during HCV replication in Huh7D cells. ( a – d ) Huh7D cells were transfected with specific plasmids for 24 h, then infected with HCV for another 24 h. ( a – c ) Protein levels were detected by Western blotting. Myc-p53 indicates p53 from ectopic expression, while Y220C-p53 represents the endogenous form in Huh7D cells. For ( b ), cells were either mock-treated or exposed to the indicated concentrations of Heclin for 12 h before harvesting. ( d ) Cell lysates were immunoprecipitated with an anti-Myc antibody, and the precipitates were analyzed by Western blotting. ( e ) Schematic diagram illustrating how HCV Core regulates proteasomal degradation of p53 mediated by MDM2 and E6AP. Each step is described in the Discussion.

Article Snippet: Scrambled (SC) small hairpin RNA (shRNA; #sc-42964) and E6AP shRNA (#sc-43742) plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Ubiquitin Proteomics, Transfection, Infection, Western Blot, Expressing, Immunoprecipitation

Journal: eLife

Article Title: UBE3A-mediated p18/LAMTOR1 ubiquitination and degradation regulate mTORC1 activity and synaptic plasticity

doi: 10.7554/eLife.37993

Figure Lengend Snippet: ( A ) Western blot analysis using anti-Ube3a, p18, or β-actin antibodies of lysates from COS-1 cells transfected with scrambled siRNA or Ube3a siRNA. Right, quantitative analysis of blots. N = 6 independent experiments, p=0.003 (unpaired, two-tailed Student's t-test). ( B ) Amino acid sequence of human p18. G2 is a myristoylation site. C3 and C4 are palmitoylation sites. K20, K31, K60, K103, K104, and K151 are potential ubiquitination sites. ( C ) Interaction between p18 and Ube3a. Lysates from COS-1 cells transfected with the indicated cDNAs in expression vectors were immunoprecipitated with an anti-Flag antibody or control IgG and probed with the indicated antibodies. The presence of Flag-p18 in precipitates was confirmed with anti-p18 and anti-Flag antibodies. ( D ) In vitro ubiquitination of p18 by recombinant Ube3a. Reaction products were analyzed by Western blots with p18, His, and ubiquitin antibodies. Note that the p18-Ub band is present only when all reaction elements are added. ( E ) Over-expression of Ube3a, but not ΔUbe3a, enhances p18 ubiquitination in COS-1 cells. His-tagged ubiquitinated proteins in cells co-transfected with HA-p18 plus empty vectors (None, but with endogenous Ube3a), wild-type Ube3a (Ube3a), or its inactive form Ube3a-C833A (ΔUbe3a) were precipitated using Talon resin and probed with anti-p18 antibodies. Ubiquitinated p18 proteins are labeled with ‘p18-(Ub)n’. Right, quantification of the relative abundance of ubiquitinated p18 (means ± SEM, p=0.009 None vs. Ube3a, p=0.022 Ube3a vs. ΔUbe3a, p=0.833 None vs. ΔUbe3a, n = 3 independent experiments, one-way ANOVA with Tukey’s post hoc analysis). ( F ) Western blot analysis using anti-Ube3a, p18, or β-actin antibodies on lysates from COS-1 cells transfected with empty vector, Ube3a, or ΔUbe3a vectors. ( G ) siRNA knockdown of Ube3a in COS-1 cells reduces p18 ubiquitination. COS-1 cells were incubated with Ube3a siRNA or scrambled control siRNA 48 hr before transfection with Flag-p18 or Flag-p18∆K and His-ubiquitin. Twenty-four hours later, ubiquitinated proteins were isolated by Co 2+ -affinity chromatography. Levels of ubiquitinated p18 protein (p18-(Ub)n, upper panel) were determined by Western blots. Levels of input proteins were also evaluated by Western blots probed with Ube3a, p18, and β-actin antibodies (lower panel). ( H ) His-ubiquitin pull-down assay performed using HA-p18 or HA-p18G2A. Upon purification, levels of ubiquitinated p18 (upper panel) were determined by Western blot analysis. Lower panel, input of Ube3a, p18, and β-actin. See also and . 10.7554/eLife.37993.004 Figure 1—source data 1. Quantitative analyses of Western blots used for and .

Article Snippet: Recombinant DNA reagent , HA-tagged wild-type Ube3a , Addgene PMID: 9497376 , 8648 , .

Techniques: Western Blot, Transfection, Two Tailed Test, Sequencing, Ubiquitin Proteomics, Expressing, Immunoprecipitation, Control, In Vitro, Recombinant, Over Expression, Labeling, Plasmid Preparation, Knockdown, Incubation, Isolation, Affinity Chromatography, Pull Down Assay, Purification

Journal: eLife

Article Title: UBE3A-mediated p18/LAMTOR1 ubiquitination and degradation regulate mTORC1 activity and synaptic plasticity

doi: 10.7554/eLife.37993

Figure Lengend Snippet: ( A ) His-ubiquitin pull-down assay performed following over-expression of Ube3a or ∆Ube3a. Upper panel: Levels of input proteins were evaluated by Western blot probed with Ube3a, p18, and β-actin antibodies. Lower panel: Levels of ubiquitin were determined by Western blot analysis. This image is paired with . ( B ) Quantitative analysis of blots in (means ± SEM, p=0.046 None vs. Ube3a, p=0.005 Ube3a vs. ∆Ube3a, p=0.195 None vs. ∆Ube3a, n = 3 independent experiments, one-way ANOVA with Tukey’s post hoc analysis). ( C ) His-ubiquitin pull-down assay performed following Ube3a siRNA treatment. Levels of ubiquitin were determined by Western blot analysis. This image is paired with . ( D ) Localization of wild-type p18 and p18G2A proteins. COS-1 cells expressing p18 or p18G2A were stained with anti-p18 antibody (red) and anti-LAMP1 antibody (green). Scale bar = 10 µm. ( E ) Western blot analysis using anti-p-4EBP1, 4EBP1, p-S6, or S6 antibodies of lysates from COS-1 cells transfected with HA-p18 or HA-p18G2A. Right, quantitative analysis of blots. N = 3 independent experiments, p=0.009 for p-4EBP1, and p=0.003 for p-S6 (unpaired, two-tailed Student's t-test). ( F ) His-ubiquitin pull-down assay performed using HA-p18 or HA-p18G2A. Levels of ubiquitin were determined by Western blot analysis. This image is paired with . ( G ) His-ubiquitin pull-down assay performed using Flag-p18 or Flag-p18 lysine mutants. Upon purification, levels of ubiquitinated p18 (p18-(Ub)n, right panel) were determined by Western blot analysis. Left panel, input of Flag and GAPDH.

Article Snippet: Recombinant DNA reagent , HA-tagged wild-type Ube3a , Addgene PMID: 9497376 , 8648 , .

Techniques: Ubiquitin Proteomics, Pull Down Assay, Over Expression, Western Blot, Expressing, Staining, Transfection, Two Tailed Test, Purification

Journal: eLife

Article Title: UBE3A-mediated p18/LAMTOR1 ubiquitination and degradation regulate mTORC1 activity and synaptic plasticity

doi: 10.7554/eLife.37993

Figure Lengend Snippet: ( A ) Left, Western blot analysis of p18 and p14 levels in crude membrane fractions (P2) of hippocampi from WT and AS mice. Right, quantitative analysis of blots. Results are expressed as % of values in WT mice and shown as means ± SEM N = 3 mice, p=0.026 (unpaired, two-tailed Student's t-test). ( B ) Interactions between Ube3a and p18 in hippocampal neuron cultures. Western blot analysis with anti-p18 and -Ube3a antibodies of immunoprecipitation performed with anti-p18 antibodies or control IgG. ( C ) Immunoprecipitation of hippocampal P2 fractions from WT and AS mice under denaturing conditions was performed with anti-ubiquitin antibodies or control IgG, and Western blots were labelled with anti-p18 antibodies. Ubiquitinated p18 proteins are indicated as ‘p18-(Ub)n’. Lower left panel: levels of input proteins were evaluated by Western blots probed with Ube3a and p18 antibodies. Lower right panel: quantification of the relative abundance of ubiquitinated p18 in hippocampus of WT and AS mice (mean ± SEM, p=0.036 compared with WT mice, n = 3 mice, Student’s t-test). ( D ) Effects of acute MG132 or bafilomycin A1 (BafA) treatment on p18 and p14 levels in hippocampus slices of WT and AS mice. Upper panel: representative Western blot images; lower panel: quantitative analysis of blots in upper panel. N = 3 independent experiments, p=0.029 WT/DMSO vs. WT/MG132, p=0.017 WT/DMSO vs. AS/DMSO, p=0.059 AS/DMSO vs. AS/MG132, two-way ANOVA with Tukey’s post-test. ( E ) Representative images of p18 in WT and AS hippocampal neurons treated with DMSO, MG132, and BafA; insets: enlarged cell bodies. Right: Quantitative analysis of images. Data are expressed as mean ± SEM. N = 3 independent experiments, p=0.013 WT/DMSO vs. WT/MG132, p=0.049 WT/DMSO vs. AS/DMSO, p=0.976 AS/DMSO vs. AS/MG132; two-way ANOVA with Tukey’s post hoc analysis. Scale bar = 20 and 10 µm in insets. See also and . 10.7554/eLife.37993.010 Figure 3—source data 1. Quantitative analyses of images and Western blots used for and .

Article Snippet: Recombinant DNA reagent , HA-tagged wild-type Ube3a , Addgene PMID: 9497376 , 8648 , .

Techniques: Western Blot, Membrane, Two Tailed Test, Immunoprecipitation, Control, Ubiquitin Proteomics

Journal: eLife

Article Title: UBE3A-mediated p18/LAMTOR1 ubiquitination and degradation regulate mTORC1 activity and synaptic plasticity

doi: 10.7554/eLife.37993

Figure Lengend Snippet: ( A ) Co-localization of p18 (red) with LAMP2 (green) in cell bodies of CA1 pyramidal neurons from WT and AS mice. Scale bar = 10 µm. ( B ) Quantification of p18-LAMP2 (n = 8 mice, p<0.001), LAMTOR4-LAMP2 (n = 6 mice, p=0.016), RagA-LAMP2 (n = 6 mice, p<0.001), and mTOR-LAMP2 (n = 8 mice, p=0.006) colocalization in cell bodies of CA1 pyramidal neurons from WT and AS mice shown in A and . Unpaired t-test. ( C ) Representative images of apical dendrites of CA1 pyramidal neurons stained with anti-mTOR (red) and -LAMP2 (green) antibodies. Arrowheads indicate puncta with dual staining. Scale bar = 5 µm. ( D ) Quantification of p18-LAMP2 (n = 8 mice, p=0.007) and mTOR-LAMP2 (n = 7 mice, p=0.011) co-localization in apical dendrites of CA1 pyramidal neurons from WT and AS mice. Unpaired t-test. ( E ) Co-localization of p-mTOR (red) with LAMP2 (green) in cell bodies of CA1 pyramidal neurons from WT and AS mice. Scale bar = 10 µm. Insets show selected fields that were magnified 10 times. ( F ) Quantification of p-mTOR-LAMP2 co-localization in cell bodies (p=0.004) and dendrites (p=0.039) of CA1 pyramidal neurons from WT and AS mice. N = 6 mice, unpaired t-test. ( G ) Homogenates from WT and AS mouse hippocampus were immunoprecipitated with an anti-RagA antibody and probed with the indicated antibodies. Right, quantification of the relative abundance of p18 bound to RagA (mean ± SEM, p=0.014, n = 3 mice, Student’s t-test). ( H ) Model proposing that the Ragulator interacts with Rag, which in turn recruits mTORC1 to be activated on lysosomes in neurons. In Ube3a-deficient neurons, increased Ragulator-Rag complex on lysosomes results in mTORC1 over-activation. See also and and . 10.7554/eLife.37993.014 Figure 4—source data 1. Quantitative analyses of images and Western blots used for and and .

Article Snippet: Recombinant DNA reagent , HA-tagged wild-type Ube3a , Addgene PMID: 9497376 , 8648 , .

Techniques: Staining, Immunoprecipitation, Activation Assay, Western Blot

Journal: eLife

Article Title: UBE3A-mediated p18/LAMTOR1 ubiquitination and degradation regulate mTORC1 activity and synaptic plasticity

doi: 10.7554/eLife.37993

Figure Lengend Snippet: ( A ) Representative images of Western blots labeled with Ube3a, p18, p-mTOR, mTOR, p-S6, S6, p-4EBP1, 4EBP1, p-PKC, and PKCα (GAPDH as a loading control). Protein lysates from cultured hippocampal neurons transfected with the indicated constructs were prepared for Western blot analysis. ( B ) Quantitative analysis of blots shown in ( A ). N = 3 independent experiments, Accell siScrambled/shScrambled vs. Accell siUbe3a/shScrambled, p=0.026 (Ube3a), p=0.001 (p18), p=0.004 (p-mTOR), p=0.006 (p–S6), p<0.001 (p-4EBP1), p=0.024 (p-PKC), p=0.007 (PKCα); Accell siScrambled/shScrambled vs. Accell siScrambled/shP18, p<0.001 (p18), p=0.008 (p-mTOR), p=0.003 (p–S6), p=0.003 (p-4EBP1), p=0.045 (p-PKC), p=0.310 (PKCα); Accell siUbe3a/shScrambled vs. Accell siUbe3a/shP18, p<0.001 (p18), p<0.001 (p-mTOR), p<0.001 (p–S6), p<0.001 (p-4EBP1), p<0.001 (p-PKC), p=0.004 (PKCα); Accell siScrambled/shP18 vs. Accell siUbe3a/shP18, p=0.034 (Ube3a); two-way ANOVA with Tukey’s post-test. ( C ) Representative images of F-actin (red) and GFP in cultured WT and AS hippocampal neurons (22 DIV) co-infected with GFP lentivirus and p18 shRNA or scrambled shRNA lentivirus. Scale bar, 20 µm (upper) or 10 µm (lower). ( D ) Quantitative analysis of images shown in ( C ). N = 9 neurons from at least three independent experiments, p<0.001, two-way ANOVA with Tukey’s post-test. See also and . 10.7554/eLife.37993.017 Figure 5—source data 1. Quantitative analyses of images and Western blots used for and .

Article Snippet: Recombinant DNA reagent , HA-tagged wild-type Ube3a , Addgene PMID: 9497376 , 8648 , .

Techniques: Western Blot, Labeling, Control, Cell Culture, Transfection, Construct, Infection, shRNA

Journal: eLife

Article Title: UBE3A-mediated p18/LAMTOR1 ubiquitination and degradation regulate mTORC1 activity and synaptic plasticity

doi: 10.7554/eLife.37993

Figure Lengend Snippet: ( A ) Quantitative analysis of the number of p18- (left, p=0.001) and PSD95-immunoreactive puncta (right, p=0.929), as well as percentage of p18 and PSD95 dually stained puncta/synapses (middle, p=0.004) in hippocampal CA1 region. N = 6 mice, unpaired t-test. These data are paired with . ( B ) The coordinates of the injection sites were (mm): AP −1.94, ML ±1.4, DV −1.35 from Bregma; AP −2.2, ML ±1.8, DV −1.5 from Bregma, in the CA1 region of hippocampus and are indicated by red circles. ( C ) Representative tile scan confocal image of GFP expression in hippocampal CA1 region 4 weeks following injection of AAV with GFP reporter gene. Scale bar = 200 μm. ( D ) Representative images of Western blots labeled with Ube3a, p18, p-mTOR, mTOR, p-S6K1, p-S6, S6, and PKCα (GAPDH as a loading control). Protein lysates from hippocampal CA1 region infected with the indicated AAV were prepared for Western blot analysis. ( E ) Effects of p18 knockdown in hippocampal CA1 region on mTOR signaling in WT and AS mice. For p-mTOR, p=0.010, WT-siScrambled vs. WT-siP18, p=0.002, WT-siScrambled vs. AS-siScrambled, p<0.001, AS-siScrambled vs. AS-siP18; For p-S6, p<0.001, WT-siScrambled vs. WT-siP18, p=0.002, WT-siScrambled vs. AS-siScrambled, p<0.001, AS-siScrambled vs. AS-siP18; For PKC, p=0.012, WT-siScrambled vs. WT-siP18, p=0.001, WT-siScrambled vs. AS-siScrambled, p<0.001, AS-siScrambled vs. AS-siP18; n = 4 mice for WT-siScrambled, WT-siP18, and AS-siScrambled, n = 3 mice for AS-siP18, two-way ANOVA with Tukey’s post-test.

Article Snippet: Recombinant DNA reagent , HA-tagged wild-type Ube3a , Addgene PMID: 9497376 , 8648 , .

Techniques: Staining, Injection, Expressing, Western Blot, Labeling, Control, Infection, Knockdown

Journal: eLife

Article Title: UBE3A-mediated p18/LAMTOR1 ubiquitination and degradation regulate mTORC1 activity and synaptic plasticity

doi: 10.7554/eLife.37993

Figure Lengend Snippet: ( A–C ) Effects of MHY1485 treatment on LTP in p18 siRNA-injected WT mice. ( A ) Slopes of fEPSPs were normalized to the average values recorded during the 10 min baseline. ( B ) Means ± SEMof fEPSPs measured 40 min after TBS in different groups. N = 3–14 slices from three to eight mice, p=0.007, unpaired t-test. ( C ) Representative Western blots showing the relative abundance of p18, p-mTOR/mTOR, and p-S6K/S6K in lysates from control siRNA (siSc) or p18 siRNA (siP18)-infected WT hippocampal slices. Slices were treated with or without MHY1485 (M). ( D,E ) Effects of Ube3a deficiency and p18 KD in the hippocampal CA1 region on Arc expression. ( D ) Representative images of CA1 pyramidal neurons stained with anti-Arc (red) and -GFP (green) antibodies. Scale bar = 50 µm (low power images) and 10 µm (high power images). ( E ) Quantitative analysis of the MFI of Arc-immunoreactivty of CA1 pyramidal neurons (means ± SEM of 3 slices from three different animals; p<0.001, WT-siScrambled vs. WT-siP18; p=0.017, WT-siScrambled vs. AS-siScrambled; p<0.001, AS-siScrambled vs. AS-siP18; p=0.016, WT-siP18 vs. AS-siP18, two-way ANOVA with Tukey’s post-hoc analysis). See also . 10.7554/eLife.37993.023 Figure 7—source data 1. Source data for .

Article Snippet: Recombinant DNA reagent , HA-tagged wild-type Ube3a , Addgene PMID: 9497376 , 8648 , .

Techniques: Injection, Western Blot, Control, Infection, Expressing, Staining

Journal: eLife

Article Title: UBE3A-mediated p18/LAMTOR1 ubiquitination and degradation regulate mTORC1 activity and synaptic plasticity

doi: 10.7554/eLife.37993

Figure Lengend Snippet: ( A ) Representative light micrograph images from Golgi-impregnated CA1 pyramidal neurons. Scale bar = 10 µm. ( B ) Quantitative analysis of mature dendritic spine (multi-head, mushroom, and stubby spines) density shown in ( A ) (means ±SEM from 10 slices). p=0.017, WT-siScrambled vs. WT-siP18; p<0.001, WT-siScrambled vs. AS-siScrambled; p<0.001, AS-siScrambled vs. AS-siP18, two-way ANOVA with Tukey’s post-test. ( C ) Representative mEPSC traces recorded in hippocampal neurons from WT and AS slices. Scale bar, 20 pA/1 s. ( D ) Quantification of mEPSC frequency (p=0.022) and amplitude (p=0.343) from WT (n = 12) and AS (n = 7) mice. Student’s t-test. ( E ) % freezing for different experimental groups in context memory (means ± SEM of 6–10 mice; p=0.043, WT-siScrambled vs. WT-siP18; p<0.001, WT-siScrambled vs. AS-siScrambled; p<0.001, AS-siScrambled vs. AS-siP18, two-way ANOVA with Tukey’s post-hoc analysis). ( F ) Model for Ube3a-mediated regulation of synaptic plasticity (see text for details). See also and . 10.7554/eLife.37993.026 Figure 8—source data 1. Source data for and .

Article Snippet: Recombinant DNA reagent , HA-tagged wild-type Ube3a , Addgene PMID: 9497376 , 8648 , .

Techniques:

Journal: eLife

Article Title: UBE3A-mediated p18/LAMTOR1 ubiquitination and degradation regulate mTORC1 activity and synaptic plasticity

doi: 10.7554/eLife.37993

Figure Lengend Snippet: Antibodies, chemicals, and plasmids used in this study

Article Snippet: Recombinant DNA reagent , HA-tagged wild-type Ube3a , Addgene PMID: 9497376 , 8648 , .

Techniques: Recombinant

Journal: eLife

Article Title: UBE3A-mediated p18/LAMTOR1 ubiquitination and degradation regulate mTORC1 activity and synaptic plasticity

doi: 10.7554/eLife.37993

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , HA-tagged wild-type Ube3a , Addgene PMID: 9497376 , 8648 , .

Techniques: Control, shRNA, Virus, Sequencing, Recombinant, Mutagenesis, Ubiquitin Proteomics, Software

Journal: Cells

Article Title: Hydrogen Peroxide Inhibits Hepatitis C Virus Replication by Downregulating Hepatitis C Virus Core Levels through E6-Associated Protein-Mediated Proteasomal Degradation

doi: 10.3390/cells13010062

Figure Lengend Snippet: H 2 O 2 downregulates HCV Core levels by upregulating E6AP levels in a p53-dependent manner. ( a – c ) Cells were transfected with the specified plasmids for 24 h and treated with H 2 O 2 for an extra 24 h, followed by Western blotting.

Article Snippet: The plasmid pCMVT N-HA-hE6AP, encoding the complete human HA-tagged E6AP , was purchased from Addgene (Watertown, MA, USA).

Techniques: Transfection, Western Blot

Journal: Cells

Article Title: Hydrogen Peroxide Inhibits Hepatitis C Virus Replication by Downregulating Hepatitis C Virus Core Levels through E6-Associated Protein-Mediated Proteasomal Degradation

doi: 10.3390/cells13010062

Figure Lengend Snippet: H 2 O 2 stimulates E6AP expression through promoter hypomethylation in the presence of p53 and HCV Core. Cells were transfected with the specified plasmids for 24 h and subsequently treated with H 2 O 2 for an extra 24 h. ( a , b ) DNMT activity from cells was determined ( n = 3). Levels of the indicated proteins were measured by Western blotting. ( c , d ) Methylation-specific PCR (MSP) was performed to determine whether the CpG sites in the E6AP promoter are unmethylated (U) or methylated (M). ( e ) Cells were treated with the specified concentration of 5-Aza-2′dC for 24 h before harvesting.

Article Snippet: The plasmid pCMVT N-HA-hE6AP, encoding the complete human HA-tagged E6AP , was purchased from Addgene (Watertown, MA, USA).

Techniques: Expressing, Transfection, Activity Assay, Western Blot, Methylation, Concentration Assay

Journal: Cells

Article Title: Hydrogen Peroxide Inhibits Hepatitis C Virus Replication by Downregulating Hepatitis C Virus Core Levels through E6-Associated Protein-Mediated Proteasomal Degradation

doi: 10.3390/cells13010062

Figure Lengend Snippet: H 2 O 2 triggers the E6AP-mediated ubiquitination and proteasomal degradation of HCV Core in a p53-dependent manner. ( a ) Cells prepared as in a,b were subjected to treatment with 50 μM cycloheximide (CHX) for the specified duration before harvesting. The quantification of each band was performed using Image J image-analysis software (NIH, USA) to determine the half-life (t 1/2 ) of HCV Core. The presented values represent the levels of HCV Core relative to the loading control (γ-tubulin). ( b ) Cells were transfected with the specified plasmids for 24 h and treated with H 2 O 2 for an extra 24 h. The transfection mixtures included the HA-Ub expression plasmid. Total HCV Core proteins in cell lysates were immunoprecipitated using an anti-HCV Core antibody and subsequently analyzed by Western blotting. The membranes were probed with antibodies against p53, HCV Core, E6AP, and HA to detect p53, HCV Core, E6AP, and HA-Ub-complexed HCV Core, respectively. Additionally, the input shows the levels of the specified proteins in the cell lysates. ( c ) Cells were either mock-treated or treated with MG132 for 4 h before harvesting.

Article Snippet: The plasmid pCMVT N-HA-hE6AP, encoding the complete human HA-tagged E6AP , was purchased from Addgene (Watertown, MA, USA).

Techniques: Software, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Cells

Article Title: Hydrogen Peroxide Inhibits Hepatitis C Virus Replication by Downregulating Hepatitis C Virus Core Levels through E6-Associated Protein-Mediated Proteasomal Degradation

doi: 10.3390/cells13010062

Figure Lengend Snippet: H 2 O 2 inhibits HCV replication in vitro through the E6AP-mediated downregulation of HCV Core levels. ( a ) Cells were transfected with the designated plasmids for 24 h and infected with HCV for an extra 24 h in the absence or presence of H 2 O 2 , followed by Western blot analysis. ( b ) The levels of HCV particles in the supernatants, as prepared in ( a ), were determined using both q-RT-PCR ( n = 4) and conventional RT-PCR. ( c ) Cells cultured in a differentiation medium (Biopredic International) for 2 weeks were infected with HCV for 24 h and treated with H 2 O 2 for an extra 24 h, followed by Western blotting. ( d ) The levels of HCV particles in the supernatants prepared in ( c ) were determined using q-RT-PCR ( n = 3) and conventional RT-PCR. ( e ) Cells were transfected with the specified plasmids for 24 h and subsequently infected with HCV for an extra 24 h, either in the absence or presence of H 2 O 2 , followed by co-IP, as described in b.

Article Snippet: The plasmid pCMVT N-HA-hE6AP, encoding the complete human HA-tagged E6AP , was purchased from Addgene (Watertown, MA, USA).

Techniques: In Vitro, Transfection, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Co-Immunoprecipitation Assay

Journal: Virology

Article Title: Binding of PDZ proteins to HPV E6 proteins does neither correlate with epidemiological risk classification nor with the immortalization of foreskin keratinocytes.

doi: 10.1016/j.virol.2009.02.018

Figure Lengend Snippet: Fig. 2. Interaction of E6 proteins with different target proteins in cell lysates. (A) Similar amounts of affinity-purified MBP or MBP-E6 proteins derived from HPV6, 11, 16, 18, 54, 56, 58, 61, 66, 70 and 82 were incubated with whole cell extracts and extensively washed. Retained proteins were eluted with amylose and then analyzed by immunoblotting with specific antibodies against p53, E6AP, hDlg or Magi1. Arrowheads indicate the presence of higher-molecular weight forms of p53 and E6AP. Experiments were repeated at least three times with independent MBP fusion protein and whole cell lysate preparations to ensure reproducibility. (B) MBP or MBP-E6 fusion proteins derived from HPV11,16,18, 56, 66 and 70 were tested for their interaction with hScrib by immunoblotting as described above.

Article Snippet: The membranes were probed with E6AP (Santa Cruz Biotechnology, H-182), p53 (Santa Cruz Biotechnology, sc126(DO-1)), hScrib (Abcam, ab6049), hDlg or Magi1 antibodies.

Techniques: Derivative Assay, Incubation, Western Blot, Molecular Weight