ha-tagged wild-type ube3a  (Addgene inc)

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

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: eLife

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

doi: 10.7554/elife.37993

Figure Lengend Snippet: Figure 1. p18 is a Ube3a substrate. (A) Western blot analysis using anti-Ube3a, p18, or b-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 Figure 1 continued on next page

Article Snippet: Goat anti-rabbit IgG AlexaFluor 633 Invitrogen Cat#A-21070 Chemicals MG132 EMD Millipore Cat#474790 Bafilomycin A1 Sigma-Aldrich Cat#B1793 MHY1485 EMD Millipore Cat#500554 Recombinant DNA HA-tagged wild-type Ube3a (Talis et al., 1998) Addgene #8648 HA-tagged Ube3a-C833A (Talis et al., 1998) Addgene #8649 HA-p18 (Bar-Peled et al., 2012) Addgene #42338 HA-p18G2A (Bar-Peled et al., 2012) Addgene #42327 Flag-p18 (Bar-Peled et al., 2012) Addgene #42331 Flag-p18DK This paper N/A Flag-p18K20R This paper N/A Flag-p18K31R This paper N/A Flag-p18K60R This paper N/A Flag-p18K103/104R This paper N/A Flag-p18K151R This paper N/A His-ubiquitin (Young et al., 2011) Addgene #31815 DOI: https://doi.org/10.7554/eLife.37993.027 Sun et al. eLife 2018;7:e37993.

Techniques: Western Blot, Transfection, Two Tailed Test, Sequencing, Ubiquitin Proteomics, Expressing

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: Figure 3. Ube3a regulates p18 levels in a proteasome-dependent manner in hippocampal neurons. (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 mm in insets. See also Figure 3—figure supplement 1 and Figure 3—source data 1. DOI: https://doi.org/10.7554/eLife.37993.008 The following source data and figure supplement are available for figure 3:

Article Snippet: Goat anti-rabbit IgG AlexaFluor 633 Invitrogen Cat#A-21070 Chemicals MG132 EMD Millipore Cat#474790 Bafilomycin A1 Sigma-Aldrich Cat#B1793 MHY1485 EMD Millipore Cat#500554 Recombinant DNA HA-tagged wild-type Ube3a (Talis et al., 1998) Addgene #8648 HA-tagged Ube3a-C833A (Talis et al., 1998) Addgene #8649 HA-p18 (Bar-Peled et al., 2012) Addgene #42338 HA-p18G2A (Bar-Peled et al., 2012) Addgene #42327 Flag-p18 (Bar-Peled et al., 2012) Addgene #42331 Flag-p18DK This paper N/A Flag-p18K20R This paper N/A Flag-p18K31R This paper N/A Flag-p18K60R This paper N/A Flag-p18K103/104R This paper N/A Flag-p18K151R This paper N/A His-ubiquitin (Young et al., 2011) Addgene #31815 DOI: https://doi.org/10.7554/eLife.37993.027 Sun et al. eLife 2018;7:e37993.

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: Figure 4. Lysosomal localization of the Ragulator-Rag complex and mTOR/p-mTOR in WT and AS mice. (A) Co-localization of p18 (red) with LAMP2 (green) in cell bodies of CA1 pyramidal neurons from WT and AS mice. Scale bar = 10 mm. (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 Figure 4—figure supplement 1A—C. 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 mm. (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 mm. 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 Figure 4—figure supplements 1 and 2 and Figure 4— source data 1. Figure 4 continued on next page

Article Snippet: Goat anti-rabbit IgG AlexaFluor 633 Invitrogen Cat#A-21070 Chemicals MG132 EMD Millipore Cat#474790 Bafilomycin A1 Sigma-Aldrich Cat#B1793 MHY1485 EMD Millipore Cat#500554 Recombinant DNA HA-tagged wild-type Ube3a (Talis et al., 1998) Addgene #8648 HA-tagged Ube3a-C833A (Talis et al., 1998) Addgene #8649 HA-p18 (Bar-Peled et al., 2012) Addgene #42338 HA-p18G2A (Bar-Peled et al., 2012) Addgene #42327 Flag-p18 (Bar-Peled et al., 2012) Addgene #42331 Flag-p18DK This paper N/A Flag-p18K20R This paper N/A Flag-p18K31R This paper N/A Flag-p18K60R This paper N/A Flag-p18K103/104R This paper N/A Flag-p18K151R This paper N/A His-ubiquitin (Young et al., 2011) Addgene #31815 DOI: https://doi.org/10.7554/eLife.37993.027 Sun et al. eLife 2018;7:e37993.

Techniques: Staining, Immunoprecipitation, Activation Assay

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: Figure 5. p18 mediates the effects of Ube3a on mTOR signaling, dendritic spine morphology, and actin polymerization. (A) Representative images of Western blots labeled with Ube3a, p18, p-mTOR, mTOR, p-S6, S6, p-4EBP1, 4EBP1, p-PKC, and PKCa (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), Figure 5 continued on next page

Article Snippet: Goat anti-rabbit IgG AlexaFluor 633 Invitrogen Cat#A-21070 Chemicals MG132 EMD Millipore Cat#474790 Bafilomycin A1 Sigma-Aldrich Cat#B1793 MHY1485 EMD Millipore Cat#500554 Recombinant DNA HA-tagged wild-type Ube3a (Talis et al., 1998) Addgene #8648 HA-tagged Ube3a-C833A (Talis et al., 1998) Addgene #8649 HA-p18 (Bar-Peled et al., 2012) Addgene #42338 HA-p18G2A (Bar-Peled et al., 2012) Addgene #42327 Flag-p18 (Bar-Peled et al., 2012) Addgene #42331 Flag-p18DK This paper N/A Flag-p18K20R This paper N/A Flag-p18K31R This paper N/A Flag-p18K60R This paper N/A Flag-p18K103/104R This paper N/A Flag-p18K151R This paper N/A His-ubiquitin (Young et al., 2011) Addgene #31815 DOI: https://doi.org/10.7554/eLife.37993.027 Sun et al. eLife 2018;7:e37993.

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

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: Figure 7. p18 KD impairs LTP in WT neurons as a result of over-inhibition of mTORC1 activity and Arc levels. (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 mm (low power images) and 10 mm (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 Figure 7—source data 1. DOI: https://doi.org/10.7554/eLife.37993.022 The following source data is available for figure 7:

Article Snippet: Goat anti-rabbit IgG AlexaFluor 633 Invitrogen Cat#A-21070 Chemicals MG132 EMD Millipore Cat#474790 Bafilomycin A1 Sigma-Aldrich Cat#B1793 MHY1485 EMD Millipore Cat#500554 Recombinant DNA HA-tagged wild-type Ube3a (Talis et al., 1998) Addgene #8648 HA-tagged Ube3a-C833A (Talis et al., 1998) Addgene #8649 HA-p18 (Bar-Peled et al., 2012) Addgene #42338 HA-p18G2A (Bar-Peled et al., 2012) Addgene #42327 Flag-p18 (Bar-Peled et al., 2012) Addgene #42331 Flag-p18DK This paper N/A Flag-p18K20R This paper N/A Flag-p18K31R This paper N/A Flag-p18K60R This paper N/A Flag-p18K103/104R This paper N/A Flag-p18K151R This paper N/A His-ubiquitin (Young et al., 2011) Addgene #31815 DOI: https://doi.org/10.7554/eLife.37993.027 Sun et al. eLife 2018;7:e37993.

Techniques: Inhibition, Activity Assay, 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: Figure 8. Effects of Ube3a deficiency and p18 KD in hippocampal CA1 region on dendritic spine morphology, mEPSCs, and fear-conditioning memory. (A) Representative light micrograph images from Golgi-impregnated CA1 pyramidal neurons. Scale bar = 10 mm. (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 Figure 8—figure supplement 1 and Figure 8—source data 1. DOI: https://doi.org/10.7554/eLife.37993.024 The following source data and figure supplement are available for figure 8:

Article Snippet: Goat anti-rabbit IgG AlexaFluor 633 Invitrogen Cat#A-21070 Chemicals MG132 EMD Millipore Cat#474790 Bafilomycin A1 Sigma-Aldrich Cat#B1793 MHY1485 EMD Millipore Cat#500554 Recombinant DNA HA-tagged wild-type Ube3a (Talis et al., 1998) Addgene #8648 HA-tagged Ube3a-C833A (Talis et al., 1998) Addgene #8649 HA-p18 (Bar-Peled et al., 2012) Addgene #42338 HA-p18G2A (Bar-Peled et al., 2012) Addgene #42327 Flag-p18 (Bar-Peled et al., 2012) Addgene #42331 Flag-p18DK This paper N/A Flag-p18K20R This paper N/A Flag-p18K31R This paper N/A Flag-p18K60R This paper N/A Flag-p18K103/104R This paper N/A Flag-p18K151R This paper N/A His-ubiquitin (Young et al., 2011) Addgene #31815 DOI: https://doi.org/10.7554/eLife.37993.027 Sun et al. eLife 2018;7:e37993.

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: Figure 1. p18 is a Ube3a substrate. (A) Western blot analysis using anti-Ube3a, p18, or b-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 Figure 1 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.37993 19 of 31 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti-p-4EBP1 Ser65 (rabbit polyclonal) Cell Signaling Technology 9451; RRID:AB_330947 (1:1000) Antibody anti-4EBP1 (rabbit monoclonal) Cell Signaling Technology 9644; RRID:AB_2097841 (1:1000) Antibody anti-p-AKT Ser473 (rabbit monoclonal) Cell Signaling Technology 4060; RRID:AB_2315049 (1:1000) Antibody anti-AKT (rabbit polyclonal) Cell Signaling Technology 9272; RRID:AB_329827 (1:1000) Antibody anti-Raptor (mouse monoclonal) EMD Millipore 05–1470; RRID:AB_10615925 (1:500) Antibody anti-Rictor (rabbit polyclonal) Bethyl Laboratories A300-459A; RRID:AB_2179967 (1:200) Antibody anti-NeuN (mouse monoclonal) EMD Millipore MAB377; RRID:AB_2298772 (1:100) Antibody anti-PSD95 (mouse monoclonal) Thermo Fisher Scientific MA1-045; RRID:AB_325399 (1:200) Antibody anti-Cathepsin B (mouse monoclonal) EMD Millipore IM27L; RRID:AB_2274848 (1:400) Antibody anti-COXIV (rabbit monoclonal) Cell Signaling Technology 4850; RRID:AB_2085424 (1:1000) Antibody anti-GFP (chicken polyclonal) Thermo Fisher Scientific A10262; RRID:AB_2534023 (1:500) Antibody anti-GAPDH (mouse monoclonal) EMD Millipore MAB374; RRID:AB_2107445 (1:1000) Antibody anti-b-actin (mouse monoclonal) Sigma-Aldrich A5441; RRID:AB_476744 (1:10,000) Antibody Goat anti-rabbit IgG IRDye 680RD LI-COR Biosciences 926–68071 (1:10,000) Antibody Goat anti-mouse IgG IRDye 800CW LI-COR Biosciences 926–32210 (1:10,000) Antibody Alexa 488- secondaries Molecular Probes (1:400) Antibody Alexa 594- or 633- secondaries Molecular Probes (1:200) Recombinant DNA reagent HA-tagged wild-type Ube3a Addgene PMID: 9497376 8648 Recombinant DNA reagent HA-tagged Ube3aC833A Addgene PMID: 9497376 8649 Recombinant DNA reagent HA-p18 Addgene PMID: 22980980 42338 Recombinant DNA reagent HA-p18G2A Addgene PMID: 22980980 42327 Recombinant DNA reagent Flag-p18 Addgene PMID: 22980980 42331 Recombinant DNA reagent Flag-p18DK This paper N/A Custom Gene Synthesis from Integrated DNA Technologies (all lysine residues in p18 mutated into arginine) Recombinant DNA reagent Flag-p18K20R This paper N/A Site-directed mutagenesis using a QuikChange II site-directed mutagenesis kit (Agilent).

Techniques: Western Blot, Transfection, Two Tailed Test, Sequencing, Ubiquitin Proteomics, Expressing

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: Figure 3. Ube3a regulates p18 levels in a proteasome-dependent manner in hippocampal neurons. (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 mm in insets. See also Figure 3—figure supplement 1 and Figure 3—source data 1. DOI: https://doi.org/10.7554/eLife.37993.008 The following source data and figure supplement are available for figure 3:

Article Snippet: DOI: https://doi.org/10.7554/eLife.37993 19 of 31 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti-p-4EBP1 Ser65 (rabbit polyclonal) Cell Signaling Technology 9451; RRID:AB_330947 (1:1000) Antibody anti-4EBP1 (rabbit monoclonal) Cell Signaling Technology 9644; RRID:AB_2097841 (1:1000) Antibody anti-p-AKT Ser473 (rabbit monoclonal) Cell Signaling Technology 4060; RRID:AB_2315049 (1:1000) Antibody anti-AKT (rabbit polyclonal) Cell Signaling Technology 9272; RRID:AB_329827 (1:1000) Antibody anti-Raptor (mouse monoclonal) EMD Millipore 05–1470; RRID:AB_10615925 (1:500) Antibody anti-Rictor (rabbit polyclonal) Bethyl Laboratories A300-459A; RRID:AB_2179967 (1:200) Antibody anti-NeuN (mouse monoclonal) EMD Millipore MAB377; RRID:AB_2298772 (1:100) Antibody anti-PSD95 (mouse monoclonal) Thermo Fisher Scientific MA1-045; RRID:AB_325399 (1:200) Antibody anti-Cathepsin B (mouse monoclonal) EMD Millipore IM27L; RRID:AB_2274848 (1:400) Antibody anti-COXIV (rabbit monoclonal) Cell Signaling Technology 4850; RRID:AB_2085424 (1:1000) Antibody anti-GFP (chicken polyclonal) Thermo Fisher Scientific A10262; RRID:AB_2534023 (1:500) Antibody anti-GAPDH (mouse monoclonal) EMD Millipore MAB374; RRID:AB_2107445 (1:1000) Antibody anti-b-actin (mouse monoclonal) Sigma-Aldrich A5441; RRID:AB_476744 (1:10,000) Antibody Goat anti-rabbit IgG IRDye 680RD LI-COR Biosciences 926–68071 (1:10,000) Antibody Goat anti-mouse IgG IRDye 800CW LI-COR Biosciences 926–32210 (1:10,000) Antibody Alexa 488- secondaries Molecular Probes (1:400) Antibody Alexa 594- or 633- secondaries Molecular Probes (1:200) Recombinant DNA reagent HA-tagged wild-type Ube3a Addgene PMID: 9497376 8648 Recombinant DNA reagent HA-tagged Ube3aC833A Addgene PMID: 9497376 8649 Recombinant DNA reagent HA-p18 Addgene PMID: 22980980 42338 Recombinant DNA reagent HA-p18G2A Addgene PMID: 22980980 42327 Recombinant DNA reagent Flag-p18 Addgene PMID: 22980980 42331 Recombinant DNA reagent Flag-p18DK This paper N/A Custom Gene Synthesis from Integrated DNA Technologies (all lysine residues in p18 mutated into arginine) Recombinant DNA reagent Flag-p18K20R This paper N/A Site-directed mutagenesis using a QuikChange II site-directed mutagenesis kit (Agilent).

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: Figure 4. Lysosomal localization of the Ragulator-Rag complex and mTOR/p-mTOR in WT and AS mice. (A) Co-localization of p18 (red) with LAMP2 (green) in cell bodies of CA1 pyramidal neurons from WT and AS mice. Scale bar = 10 mm. (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 Figure 4—figure supplement 1A—C. 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 mm. (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 mm. 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 Figure 4—figure supplements 1 and 2 and Figure 4— source data 1. Figure 4 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.37993 19 of 31 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti-p-4EBP1 Ser65 (rabbit polyclonal) Cell Signaling Technology 9451; RRID:AB_330947 (1:1000) Antibody anti-4EBP1 (rabbit monoclonal) Cell Signaling Technology 9644; RRID:AB_2097841 (1:1000) Antibody anti-p-AKT Ser473 (rabbit monoclonal) Cell Signaling Technology 4060; RRID:AB_2315049 (1:1000) Antibody anti-AKT (rabbit polyclonal) Cell Signaling Technology 9272; RRID:AB_329827 (1:1000) Antibody anti-Raptor (mouse monoclonal) EMD Millipore 05–1470; RRID:AB_10615925 (1:500) Antibody anti-Rictor (rabbit polyclonal) Bethyl Laboratories A300-459A; RRID:AB_2179967 (1:200) Antibody anti-NeuN (mouse monoclonal) EMD Millipore MAB377; RRID:AB_2298772 (1:100) Antibody anti-PSD95 (mouse monoclonal) Thermo Fisher Scientific MA1-045; RRID:AB_325399 (1:200) Antibody anti-Cathepsin B (mouse monoclonal) EMD Millipore IM27L; RRID:AB_2274848 (1:400) Antibody anti-COXIV (rabbit monoclonal) Cell Signaling Technology 4850; RRID:AB_2085424 (1:1000) Antibody anti-GFP (chicken polyclonal) Thermo Fisher Scientific A10262; RRID:AB_2534023 (1:500) Antibody anti-GAPDH (mouse monoclonal) EMD Millipore MAB374; RRID:AB_2107445 (1:1000) Antibody anti-b-actin (mouse monoclonal) Sigma-Aldrich A5441; RRID:AB_476744 (1:10,000) Antibody Goat anti-rabbit IgG IRDye 680RD LI-COR Biosciences 926–68071 (1:10,000) Antibody Goat anti-mouse IgG IRDye 800CW LI-COR Biosciences 926–32210 (1:10,000) Antibody Alexa 488- secondaries Molecular Probes (1:400) Antibody Alexa 594- or 633- secondaries Molecular Probes (1:200) Recombinant DNA reagent HA-tagged wild-type Ube3a Addgene PMID: 9497376 8648 Recombinant DNA reagent HA-tagged Ube3aC833A Addgene PMID: 9497376 8649 Recombinant DNA reagent HA-p18 Addgene PMID: 22980980 42338 Recombinant DNA reagent HA-p18G2A Addgene PMID: 22980980 42327 Recombinant DNA reagent Flag-p18 Addgene PMID: 22980980 42331 Recombinant DNA reagent Flag-p18DK This paper N/A Custom Gene Synthesis from Integrated DNA Technologies (all lysine residues in p18 mutated into arginine) Recombinant DNA reagent Flag-p18K20R This paper N/A Site-directed mutagenesis using a QuikChange II site-directed mutagenesis kit (Agilent).

Techniques: Staining, Immunoprecipitation, Activation Assay

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: Figure 5. p18 mediates the effects of Ube3a on mTOR signaling, dendritic spine morphology, and actin polymerization. (A) Representative images of Western blots labeled with Ube3a, p18, p-mTOR, mTOR, p-S6, S6, p-4EBP1, 4EBP1, p-PKC, and PKCa (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), Figure 5 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.37993 19 of 31 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti-p-4EBP1 Ser65 (rabbit polyclonal) Cell Signaling Technology 9451; RRID:AB_330947 (1:1000) Antibody anti-4EBP1 (rabbit monoclonal) Cell Signaling Technology 9644; RRID:AB_2097841 (1:1000) Antibody anti-p-AKT Ser473 (rabbit monoclonal) Cell Signaling Technology 4060; RRID:AB_2315049 (1:1000) Antibody anti-AKT (rabbit polyclonal) Cell Signaling Technology 9272; RRID:AB_329827 (1:1000) Antibody anti-Raptor (mouse monoclonal) EMD Millipore 05–1470; RRID:AB_10615925 (1:500) Antibody anti-Rictor (rabbit polyclonal) Bethyl Laboratories A300-459A; RRID:AB_2179967 (1:200) Antibody anti-NeuN (mouse monoclonal) EMD Millipore MAB377; RRID:AB_2298772 (1:100) Antibody anti-PSD95 (mouse monoclonal) Thermo Fisher Scientific MA1-045; RRID:AB_325399 (1:200) Antibody anti-Cathepsin B (mouse monoclonal) EMD Millipore IM27L; RRID:AB_2274848 (1:400) Antibody anti-COXIV (rabbit monoclonal) Cell Signaling Technology 4850; RRID:AB_2085424 (1:1000) Antibody anti-GFP (chicken polyclonal) Thermo Fisher Scientific A10262; RRID:AB_2534023 (1:500) Antibody anti-GAPDH (mouse monoclonal) EMD Millipore MAB374; RRID:AB_2107445 (1:1000) Antibody anti-b-actin (mouse monoclonal) Sigma-Aldrich A5441; RRID:AB_476744 (1:10,000) Antibody Goat anti-rabbit IgG IRDye 680RD LI-COR Biosciences 926–68071 (1:10,000) Antibody Goat anti-mouse IgG IRDye 800CW LI-COR Biosciences 926–32210 (1:10,000) Antibody Alexa 488- secondaries Molecular Probes (1:400) Antibody Alexa 594- or 633- secondaries Molecular Probes (1:200) Recombinant DNA reagent HA-tagged wild-type Ube3a Addgene PMID: 9497376 8648 Recombinant DNA reagent HA-tagged Ube3aC833A Addgene PMID: 9497376 8649 Recombinant DNA reagent HA-p18 Addgene PMID: 22980980 42338 Recombinant DNA reagent HA-p18G2A Addgene PMID: 22980980 42327 Recombinant DNA reagent Flag-p18 Addgene PMID: 22980980 42331 Recombinant DNA reagent Flag-p18DK This paper N/A Custom Gene Synthesis from Integrated DNA Technologies (all lysine residues in p18 mutated into arginine) Recombinant DNA reagent Flag-p18K20R This paper N/A Site-directed mutagenesis using a QuikChange II site-directed mutagenesis kit (Agilent).

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

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: Figure 7. p18 KD impairs LTP in WT neurons as a result of over-inhibition of mTORC1 activity and Arc levels. (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 mm (low power images) and 10 mm (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 Figure 7—source data 1. DOI: https://doi.org/10.7554/eLife.37993.022 The following source data is available for figure 7:

Article Snippet: DOI: https://doi.org/10.7554/eLife.37993 19 of 31 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti-p-4EBP1 Ser65 (rabbit polyclonal) Cell Signaling Technology 9451; RRID:AB_330947 (1:1000) Antibody anti-4EBP1 (rabbit monoclonal) Cell Signaling Technology 9644; RRID:AB_2097841 (1:1000) Antibody anti-p-AKT Ser473 (rabbit monoclonal) Cell Signaling Technology 4060; RRID:AB_2315049 (1:1000) Antibody anti-AKT (rabbit polyclonal) Cell Signaling Technology 9272; RRID:AB_329827 (1:1000) Antibody anti-Raptor (mouse monoclonal) EMD Millipore 05–1470; RRID:AB_10615925 (1:500) Antibody anti-Rictor (rabbit polyclonal) Bethyl Laboratories A300-459A; RRID:AB_2179967 (1:200) Antibody anti-NeuN (mouse monoclonal) EMD Millipore MAB377; RRID:AB_2298772 (1:100) Antibody anti-PSD95 (mouse monoclonal) Thermo Fisher Scientific MA1-045; RRID:AB_325399 (1:200) Antibody anti-Cathepsin B (mouse monoclonal) EMD Millipore IM27L; RRID:AB_2274848 (1:400) Antibody anti-COXIV (rabbit monoclonal) Cell Signaling Technology 4850; RRID:AB_2085424 (1:1000) Antibody anti-GFP (chicken polyclonal) Thermo Fisher Scientific A10262; RRID:AB_2534023 (1:500) Antibody anti-GAPDH (mouse monoclonal) EMD Millipore MAB374; RRID:AB_2107445 (1:1000) Antibody anti-b-actin (mouse monoclonal) Sigma-Aldrich A5441; RRID:AB_476744 (1:10,000) Antibody Goat anti-rabbit IgG IRDye 680RD LI-COR Biosciences 926–68071 (1:10,000) Antibody Goat anti-mouse IgG IRDye 800CW LI-COR Biosciences 926–32210 (1:10,000) Antibody Alexa 488- secondaries Molecular Probes (1:400) Antibody Alexa 594- or 633- secondaries Molecular Probes (1:200) Recombinant DNA reagent HA-tagged wild-type Ube3a Addgene PMID: 9497376 8648 Recombinant DNA reagent HA-tagged Ube3aC833A Addgene PMID: 9497376 8649 Recombinant DNA reagent HA-p18 Addgene PMID: 22980980 42338 Recombinant DNA reagent HA-p18G2A Addgene PMID: 22980980 42327 Recombinant DNA reagent Flag-p18 Addgene PMID: 22980980 42331 Recombinant DNA reagent Flag-p18DK This paper N/A Custom Gene Synthesis from Integrated DNA Technologies (all lysine residues in p18 mutated into arginine) Recombinant DNA reagent Flag-p18K20R This paper N/A Site-directed mutagenesis using a QuikChange II site-directed mutagenesis kit (Agilent).

Techniques: Inhibition, Activity Assay, 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: Figure 8. Effects of Ube3a deficiency and p18 KD in hippocampal CA1 region on dendritic spine morphology, mEPSCs, and fear-conditioning memory. (A) Representative light micrograph images from Golgi-impregnated CA1 pyramidal neurons. Scale bar = 10 mm. (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 Figure 8—figure supplement 1 and Figure 8—source data 1. DOI: https://doi.org/10.7554/eLife.37993.024 The following source data and figure supplement are available for figure 8:

Article Snippet: DOI: https://doi.org/10.7554/eLife.37993 19 of 31 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti-p-4EBP1 Ser65 (rabbit polyclonal) Cell Signaling Technology 9451; RRID:AB_330947 (1:1000) Antibody anti-4EBP1 (rabbit monoclonal) Cell Signaling Technology 9644; RRID:AB_2097841 (1:1000) Antibody anti-p-AKT Ser473 (rabbit monoclonal) Cell Signaling Technology 4060; RRID:AB_2315049 (1:1000) Antibody anti-AKT (rabbit polyclonal) Cell Signaling Technology 9272; RRID:AB_329827 (1:1000) Antibody anti-Raptor (mouse monoclonal) EMD Millipore 05–1470; RRID:AB_10615925 (1:500) Antibody anti-Rictor (rabbit polyclonal) Bethyl Laboratories A300-459A; RRID:AB_2179967 (1:200) Antibody anti-NeuN (mouse monoclonal) EMD Millipore MAB377; RRID:AB_2298772 (1:100) Antibody anti-PSD95 (mouse monoclonal) Thermo Fisher Scientific MA1-045; RRID:AB_325399 (1:200) Antibody anti-Cathepsin B (mouse monoclonal) EMD Millipore IM27L; RRID:AB_2274848 (1:400) Antibody anti-COXIV (rabbit monoclonal) Cell Signaling Technology 4850; RRID:AB_2085424 (1:1000) Antibody anti-GFP (chicken polyclonal) Thermo Fisher Scientific A10262; RRID:AB_2534023 (1:500) Antibody anti-GAPDH (mouse monoclonal) EMD Millipore MAB374; RRID:AB_2107445 (1:1000) Antibody anti-b-actin (mouse monoclonal) Sigma-Aldrich A5441; RRID:AB_476744 (1:10,000) Antibody Goat anti-rabbit IgG IRDye 680RD LI-COR Biosciences 926–68071 (1:10,000) Antibody Goat anti-mouse IgG IRDye 800CW LI-COR Biosciences 926–32210 (1:10,000) Antibody Alexa 488- secondaries Molecular Probes (1:400) Antibody Alexa 594- or 633- secondaries Molecular Probes (1:200) Recombinant DNA reagent HA-tagged wild-type Ube3a Addgene PMID: 9497376 8648 Recombinant DNA reagent HA-tagged Ube3aC833A Addgene PMID: 9497376 8649 Recombinant DNA reagent HA-p18 Addgene PMID: 22980980 42338 Recombinant DNA reagent HA-p18G2A Addgene PMID: 22980980 42327 Recombinant DNA reagent Flag-p18 Addgene PMID: 22980980 42331 Recombinant DNA reagent Flag-p18DK This paper N/A Custom Gene Synthesis from Integrated DNA Technologies (all lysine residues in p18 mutated into arginine) Recombinant DNA reagent Flag-p18K20R This paper N/A Site-directed mutagenesis using a QuikChange II site-directed mutagenesis kit (Agilent).

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: 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