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