Journal: PLoS ONE

Article Title: Adeno-Associated Virus-Mediated Rescue of the Cognitive Defects in a Mouse Model for Angelman Syndrome

doi: 10.1371/journal.pone.0027221

Figure Lengend Snippet: (A) Representative coronal slices through the hippocampus from each group stained for E6-AP. (B) Quantitative analysis of the IHC revealed a significant increase in E6-AP expression in the WT and AS-TR2-UBE3A mice compared to the AS TR2-GFP group, while there was no significant change between the WT and AS TR2-UBE3A mice. Results shown represent the mean with standard error.

Article Snippet: Virus was generated by cotransfection of the UBE3A plasmid with the helper plasmids pXX6 and pAAV9 in HEK293 cells (ATCC, Manassas, VA).

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: Adeno-Associated Virus-Mediated Rescue of the Cognitive Defects in a Mouse Model for Angelman Syndrome

doi: 10.1371/journal.pone.0027221

Figure Lengend Snippet: (A) There were no differences during the training phase of fear conditioning, indicating that all groups of mice were capable of freezing to the same extent. (B) AS TR2-GFP mice show significant deficits in contextual fear conditioning when assessed 24 h after training. AS TR2-UBE3A mice, however, froze at the same rate as the wildtype mice. Results shown represent the mean with standard error.

Article Snippet: Virus was generated by cotransfection of the UBE3A plasmid with the helper plasmids pXX6 and pAAV9 in HEK293 cells (ATCC, Manassas, VA).

Techniques:

Journal: PLoS ONE

Article Title: Adeno-Associated Virus-Mediated Rescue of the Cognitive Defects in a Mouse Model for Angelman Syndrome

doi: 10.1371/journal.pone.0027221

Figure Lengend Snippet: (A) Escape latency to reach the platform during 5 days of training in Morris water maze. The only significant differences seen were an increase in latency for the AS TR2-GFP mice on day 3 and a decrease in latency for wildtype mice on day 4 compared to the other two groups (2-way ANOVA Bonferroni: Interaction [F(8,100) = 1.01, P>0.05 ]; Treatment [F(2,100) = 5.30, P<0.05 ]; Time [F(4,100) = 53.49, P<0.0001 ]; Matching [F(25,100) = 5.37, P<0.0001 ]). The target platform is indicated by the black circles. (B) Quantification of the number of platform crossings in the target (T), opposite (O), right (R), and left (L) quadrants during the probe trial of the Morris water maze 24 hours after training indicate no significant differences among any of the groups in comparing target platform crossings to opposite platform crossings (ANOVA Tukey WT: [F(3,35) = 9.546, P<0.0005 ]; AS TR2-GFP: [F(3,35) = 3.186, P<0.01 ]; AS TR2-UBE3A: [F(3,39) = 2.814, P<0.05 ]). All three groups learned the platform location based on a spatial bias as indicated by the time spent in the target quadrants ( 24 h ANOVA Tukey [F(2,26) = 1.027, P>0.05 ]) (C) A probe test 72 hours after training indicate that the WT and AS TR2-UBE3A groups had significantly more target platform crossings compared to the number of opposite platform crossings (ANOVA Tukey WT: [F(3,35) = 6.086, P<0.005 ]; AS TR2-GFP: [F(3,35) = 0.5650, P>0.05 ]; AS TR2-UBE3A: [F(3,39) = 2.679, P<0.05 ]), but this improvement was not spatially biased as seen by the time spent in the target quadrant ( 72 h ANOVA Tukey [F(2,25) = 5.067, P<0.02 ]). Results shown represent the mean with standard error.

Article Snippet: Virus was generated by cotransfection of the UBE3A plasmid with the helper plasmids pXX6 and pAAV9 in HEK293 cells (ATCC, Manassas, VA).

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: ( 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: HA-tagged wild-type Ube3a , ( ) , Addgene #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: HA-tagged wild-type Ube3a , ( ) , Addgene #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: HA-tagged wild-type Ube3a , ( ) , Addgene #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: HA-tagged wild-type Ube3a , ( ) , Addgene #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: HA-tagged wild-type Ube3a , ( ) , Addgene #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: HA-tagged wild-type Ube3a , ( ) , Addgene #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: HA-tagged wild-type Ube3a , ( ) , Addgene #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: HA-tagged wild-type Ube3a , ( ) , Addgene #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: HA-tagged wild-type Ube3a , ( ) , Addgene #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: HA-tagged wild-type Ube3a , ( ) , Addgene #8648.

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

Journal: PLoS ONE

Article Title: GRIM-19 Disrupts E6/E6AP Complex to Rescue p53 and Induce Apoptosis in Cervical Cancers

doi: 10.1371/journal.pone.0022065

Figure Lengend Snippet: ( A ) Co-immunoprecipitation assays were performed to determine the interaction between GRIM-19 with E6AP in vivo . The cell lysates from HeLa cells were immunoprecipitated with normal IgG and anti-GRIM-19 antibodies and Western blotted with anti-E6AP. Input (preIP) lane represents 10% of the extract used in the immunoprecipitation reaction. HC = IgG heavy chain. ( B ) GST pull-down experiments were performed to examine the interaction of His-tagged E6AP deletions with GST-fused GRIM-19 protein in vitro . ( C ) Schematic diagram of E6AP indicating various functional domains including the binding sites for GRIM-19. ( D ) The interaction of GST-GRIM-19 deletions with E6AP. * indicates the position of the band with correct molecular size. ( E ) Co-immunoprecipitation assays were performed to determine the interaction between GRIM-19 with 18E6. The cell lysates from HeLa cells transfected with the p18E6-Flag were subjected to IP with the indicated antibodies. Input (preIP) lane represents 10% of the extract used in the immunoprecipitation reaction. LC = IgG light chain. ( F ) The interaction of GST-GRIM-19 deletions with 18E6. * indicates the position of the band with correct molecular size. ( G ) Deletion mapping of the E6 or E6AP binding sites on the GRIM-19.

Article Snippet: To construct the bacterial recombinant plasmid expressing His-tagged wild-type E6AP protein, the coding sequence of E6AP was PCR amplified using pCMV6-XL5-E6AP-III (Origene, SC120518, USA,) as a template with the following primer pair: a sense primer with an NdeI restriction enzyme site (underlined): 5′-GGGAATTC CATATG GCCACAGCTTGTAAAAGA TCAGG -3′ ; and an antisense primer with a HindIII restriction enzyme site (underlined): 5′-CCC AAGCTT CA GCATGCCAAATCCTTTGG -3′ .

Techniques: Immunoprecipitation, In Vivo, Western Blot, In Vitro, Functional Assay, Binding Assay, Transfection

Journal: PLoS ONE

Article Title: GRIM-19 Disrupts E6/E6AP Complex to Rescue p53 and Induce Apoptosis in Cervical Cancers

doi: 10.1371/journal.pone.0022065

Figure Lengend Snippet: ( A ) Competitive assays were performed to analyze the binding of 18E6 to GST-GRIM-19 fusion protein in presence of increasing amounts of E6AP-Δ3 ranging from 0–6 µg. ( B ) Pull-down experiments to determine the binding of 18E6 to GST-GRIM-19 proteins. Where indicated E6AP-Δ2 proteins (10 ug) were added into GST Pull-down reaction. ( C ) GRIM-19 augmented E6AP degradation in vivo . Before harvesting the cells were treated with MG132 for 4 h, and immunoprecipitation with E6AP antibody was performed. Western blotting of the IP products was using ubiquitin antibody. GAPDH antibodies were used to determine the comparable loading. ( D ) In vitro E6AP ubiquitination assay. Human recombinant ubiquitin, E1, E2 (UbcH5c), batereria-expressed and purified GST and GTS-GRIM-19, E6AP (wild-type or catalytically inactive mutant CA) from wheat germ extract were mixed for in vitro E6AP ubiquitination assay and immunoblotted with ubiquitin antibody. ( E ) Whole cell lysates from HeLa cells expressing the indicated expression plasmids were Western blotted with the indicated antibodies.

Article Snippet: To construct the bacterial recombinant plasmid expressing His-tagged wild-type E6AP protein, the coding sequence of E6AP was PCR amplified using pCMV6-XL5-E6AP-III (Origene, SC120518, USA,) as a template with the following primer pair: a sense primer with an NdeI restriction enzyme site (underlined): 5′-GGGAATTC CATATG GCCACAGCTTGTAAAAGA TCAGG -3′ ; and an antisense primer with a HindIII restriction enzyme site (underlined): 5′-CCC AAGCTT CA GCATGCCAAATCCTTTGG -3′ .

Techniques: Binding Assay, In Vivo, Immunoprecipitation, Western Blot, In Vitro, Ubiquitin Assay, Recombinant, Purification, Mutagenesis, Expressing

Journal: PLoS ONE

Article Title: GRIM-19 Disrupts E6/E6AP Complex to Rescue p53 and Induce Apoptosis in Cervical Cancers

doi: 10.1371/journal.pone.0022065

Figure Lengend Snippet: ( A ) An MTT assay was performed in the indicated cells. MTT assay was performed as in . Each data point represents the mean ± SE of 8 samples. ( B ) The morphological characteristics of HeLa/pCon and HeLa/pG19 cells were examined by transmission electron microscopy. Chromatin condensation, expansion and widened nuclear membrane gaps, vague nuclear membrane structure, fractures of nuclear membrane and endoplasmic reticulum expansion were indicated with arrows. ER, endoplasmic reticulum; NM, nuclear membrane. ( C ) The full length and cleaved form of caspase-3 and PARP in HeLa/pCon and HeLa/pG19 cells were determined by Western blot analyses. ( D ) HeLa/Con and HeLa/G19 cells were transplanted into 6-week-old female athymic nude mice (10 mice for each cell line) and grown for 6 weeks. Tumors were harvested, and weights were measured. The data present the mean of 10 tumors in each group. ( E ) The expression of GRIM-19, p53, p21, PUMA, p27 and E6AP in the tumors derived from mice as determined by Western blot analyses, and the representative results are presented. ( F ) A model for the collaboration between GRIM-19 and p53. When GRIM-19 is present in high levels, it interacts with the E6/E6AP complex, promotes their ubiquitination, thus, preventing p53 degradation. The loss of GRIM-19 allows the attack of E6/E6AP complex on p53 and its degradation through the proteasome.

Article Snippet: To construct the bacterial recombinant plasmid expressing His-tagged wild-type E6AP protein, the coding sequence of E6AP was PCR amplified using pCMV6-XL5-E6AP-III (Origene, SC120518, USA,) as a template with the following primer pair: a sense primer with an NdeI restriction enzyme site (underlined): 5′-GGGAATTC CATATG GCCACAGCTTGTAAAAGA TCAGG -3′ ; and an antisense primer with a HindIII restriction enzyme site (underlined): 5′-CCC AAGCTT CA GCATGCCAAATCCTTTGG -3′ .

Techniques: MTT Assay, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Derivative Assay