Journal: The Journal of Cell Biology

Article Title: Ecm29-mediated proteasomal distribution modulates excitatory GABA responses in the developing brain

doi: 10.1083/jcb.201903033

Figure Lengend Snippet: Asymmetric proteasome distribution requires AIS integrity. (A and B) Either AnkG knockdown or induction of ischemia-like conditions (OGD) impairs AIS structure and proteasome distribution in cultures of mouse neurons on 5 DIV. Images of hippocampal neurons transfected with scrambled control (SC-siRNA) or AnkG-siRNA 30 min after plating, followed by immunostaining for the AIS marker AnkG (red), proteasome subunit Rpt5 (green, A), or the proteasome adaptor Ecm29 (green, B) at 5 DIV. Rightmost panels show regions of interest (dashed boxes) of the AIS represented at higher magnification, with Rpt5 (A) or Ecm29 (B) staining intensity indicated by a linear pseudocolor scale. Plots at right reveal a more uniform distribution (reflected by flatter slopes) of Rpt5 (blue trace, A) and Ecm29 (blue trace, B) across the AIS region and distal axon following AnkG knockdown or after 30 min of OGD conditions. Scale bar, 20 µm. Traces of neuron-specific class III β-tubulin (Tuj1) show that the microtubule bundle was not altered following AnkG knockdown. Traces represent averages of >50 cells per group from three independent experiments. m , slope of the intensity profile of Rpt5 or Ecm29 across the AIS. (C and D) MV151-labeled proteasomes are retained in the AIS region. (C) Top: Representative image showing a 5-DIV cortical neuron stained with neurofascin antibody and MV151 to visualize AIS position and proteasome movement, respectively, during live-cell imaging. Middle: Schematic showing position of the AIS (neurofascin-positive segment) and distal axon regions, defined as the axonal segment 60–80 µm from cell body. Bottom: Representative kymographs (2 s/frame; 180 s) of MV151-labeled proteasomes in axons of 5-DIV neurons following treatments indicated at left. Corresponding color-coded trajectories plotted for a subset of MV151-labeled proteasomes (blue lines, particles moving through AIS region; black lines, particles blocked or static in the proximal axon). Static particles outside the AIS are not marked. (D) Summary histograms showing that either AnkG knockdown by siRNA-AnkG or ischemia-like conditions (OGD for 30 min) significantly increase the percentage of MV151-labeled proteasomes across the AIS (denoted as “moving through”) in 5- or 6-DIV neurons. Note that treatment with the actin filament–depolymerizing agent latrunculin A (Lat A; 1 µM for 6 h) had no effect on proteasome transport, whereas the microtubule-disrupting agent nocodazole (Noco; 1 µM for 3 h) or Ecm29 KO (Ecm29 −/− ) abolished all transport. con, Control Ecm29 +/+ group; # , no transport. Mean ± SEM, n = 20 cells per group from three independent experiments, 10–15 puncta each cell; *, P < 0.05; **, P < 0.01; ns, not significant compared with control group by unpaired t test. Scale bar, 20 µm. (E) Quantification of proteins at the AIS relative to those in the distal axon region in experiments conducted as described in C and D, except that 5-DIV cortical neurons were immunostained for Ecm29, Rpt5, and AnkG. Summary histograms show an even distribution of Ecm29 and Rpt5 over the AIS and distal axonal region in AnkG knockdown (AnkG-siRNA) neurons or OGD conditions, as indicated. Data represent mean ± SEM; n > 4 independent experiments, >50 cells per group; *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant compared with control group by unpaired t test (Ecm29 and Rpt5 panels), or by one-way ANOVA with Dunnett’s multiple comparison test (AnkG panel).

Article Snippet: AnkG siRNA constructs (Accell SMARTpool Ank3 siRNAs), a mixture of four siRNAs (5′-CUG​GCU​UCC​UUU​ACG​UUU​U-3′, 5′-CUA​UGA​AUU​GUA​GAA​GUA​U-3′, 5′-CGC​UUA​UUU​AAA​UGU​CUU​A-3′, and 5′-UCA​UAA​UUA​AGG​AUU​UGU​A-3′), proteasome subunit Rpt1 siRNA constructs (5′-UUG​UUA​ACC​UUG​GAA​UCG​A-3′, 5′-GUC​GUA​UGC​CAA​AUU​UAG​U-3′, 5′-GCA​AGA​UGU​ACC​AAG​AUA​A-3′, and 5′-GCG​GAG​AUU​AGA​AGC​GUU​U-3′), and a scrambled siRNA (Acell Non-targeting siRNA #1: 5′-UGG​UUU​ACA​UGU​CGA​CUA​A-3′) were purchased from Dharmacon.

Techniques: Transfection, Immunostaining, Marker, Staining, Labeling, Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: Ecm29-mediated proteasomal distribution modulates excitatory GABA responses in the developing brain

doi: 10.1083/jcb.201903033

Figure Lengend Snippet: Proteasome retention and local protein degradation in the AIS require Ecm29 interaction with AnkG. (A) In vivo protein binding assays in HEK293T cells transfected with a plasmid encoding FLAG-tagged Ecm29 1–1,840 (FLAG-Ecm29 FL ) and plasmids encoding 190AnkG-GFP (AnkG-GFP) or control GFP vectors, as indicated. Cell lysates were IP by FLAG M2 antibodies and blotted with indicated antibodies. Histograms at right show relative protein levels based on immunoblotting of GFP, Arp1, Rpt1, and KLC1 coIP by FLAG antibodies (±SEM; n = 4 independent experiments; *, P < 0.05; ns, not significant, by t test). (B) cAMP increases facilitate Ecm29–AnkG interaction. (B1) Schematic showing design of AIS-mimicking ratiometric FRET sensors. AIS molecular dock: NF-186. Donor molecule: 190AnkG-GFP. Acceptor molecule: Ecm29ΔC-mCherry or Ecm29ΔN-mCherry. (B2) Time-lapse FRET images of HEK293T cells cotransfected with plasmids encoding 190AnkG-GFP, NF-186-HA, and N- or C-terminal Ecm29-mCherry (Ecm29ΔC-mCherry or Ecm29ΔN-mCherry) in the presence of 20 µM forskolin, with or without 30 min of pretreatment with the PKA inhibitor KT5720, as indicated. White dashed line passes through the center of the cell, with points a and b as intersection points. Scale bar, 10 µm. (B3) Quantitative measurement of FRET signals (±SEM, n ≥ 13 cells for each group) at different times before and after addition of forskolin (20 µM) in experiments as described in B. Arrow in right panel denotes forskolin addition. Norm., normalized. (C) Ecm29 expression is required for proteasome association with the detergent-resistant AIS structure. (C1) Representative images of 7-DIV hippocampal neurons transfected with SC-shRNA or AnkG-targeting siRNA (AnkG-siRNA) at 3 DIV, with or without nocodazole (Noco; 1 µM for 3 h) or latrunculin A (Lat A; 1 µM for 6 h), followed by detergent extraction with 0.1% Triton X-100 at 37°C for 2 min before immunostaining for the AIS marker AnkG/Trim46 (red, merge panels), proteasome subunit Rpt1/Rpt5 (green, merge panels), and the proteasome adaptor Ecm29 (green, merge panels). (C2) Quantitative measurement of immunostaining intensities (±SEM, n = 15–35 cells) along AIS segments. (D) Local protein turnover at the AIS requires Ecm29 expression and proteasome activity. (D1) Images of 7-DIV hippocampal neurons expressing an AIS-located protein degradation reporter, Na v II-III–tagged GFPu, showing more durable GFPu accumulation after MG132 pretreatment (2.5 µM, 30 min) or shRNA-mediated knockdown of Rpt-1 or Ecm29 expression, as indicated. Scale bar, 15 µm. (D2) Quantitative measurement of fluorescence intensities in the AIS (top panel) and soma (bottom panel) at different time points based on all experiments performed as described in D1 (±SEM, n > 6 cells for each group, one-way ANOVA followed by Dunnett’s multiple comparison test. *, P < 0.05; ns, not significant).

Article Snippet: AnkG siRNA constructs (Accell SMARTpool Ank3 siRNAs), a mixture of four siRNAs (5′-CUG​GCU​UCC​UUU​ACG​UUU​U-3′, 5′-CUA​UGA​AUU​GUA​GAA​GUA​U-3′, 5′-CGC​UUA​UUU​AAA​UGU​CUU​A-3′, and 5′-UCA​UAA​UUA​AGG​AUU​UGU​A-3′), proteasome subunit Rpt1 siRNA constructs (5′-UUG​UUA​ACC​UUG​GAA​UCG​A-3′, 5′-GUC​GUA​UGC​CAA​AUU​UAG​U-3′, 5′-GCA​AGA​UGU​ACC​AAG​AUA​A-3′, and 5′-GCG​GAG​AUU​AGA​AGC​GUU​U-3′), and a scrambled siRNA (Acell Non-targeting siRNA #1: 5′-UGG​UUU​ACA​UGU​CGA​CUA​A-3′) were purchased from Dharmacon.

Techniques: In Vivo, Protein Binding, Transfection, Plasmid Preparation, Western Blot, Expressing, shRNA, Immunostaining, Marker, Activity Assay, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Ecm29-mediated proteasomal distribution modulates excitatory GABA responses in the developing brain

doi: 10.1083/jcb.201903033

Figure Lengend Snippet: Proteasome and NKCC1 activities modulate AIS dynamics via Ecm29. (A) Ecm29 loss alters developmental AIS dynamics. (A1) Representative images of cortical neurons treated with or without 10 µM BUM and immunostained with AnkG antibodies at indicated time points. Scale bar, 20 µm. (A2) Scatter plot showing shorter distances from cell bodies of AIS start positions (upper panel) in Ecm29 KO (Ecm29 −/− ) cortical neurons relative to wild-type (Ecm29 +/+ ) neurons in ∼5–7-DIV cultures. AIS start and end positions were estimated based on positions of 50% peak AnkG intensity at both tails, with the distance between these positions defining AIS length (lower panel). *, P < 0.05; ****, P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons post hoc test. (A3) Cumulative probability plot showing that NKCC1 inhibition by 10 µM BUM treatment (+BUM; added at 4 DIV) restored the abnormally accelerated proximal shift in AIS position (upper panel) in 7-DIV Ecm29 KO neurons. Data from experiments similar to those described in A1 and A2. ****, P < 0.0001; ns, not significant, two-way ANOVA with Dunnett’s post hoc test. (B) Blockade of proteasome activity causes an early proximal shift in AIS position in 7-DIV cortical neurons. (B1) Representative images of 7-DIV cortical neurons treated with or without proteasome inhibitors (MG132 or lactacystin; 2.5 µM, 8 h), and ectopically expressing SC-siRNA or Rpt1 targeting-siRNA (Rpt1-siRNA A-D), and/or control GFP or Ecm29-IRES-GFP, followed by immunostaining with AnkG antibodies (red) or Rpt1 antibody (gray). Scale bars, 20 μm. (B2 and B3) Averaged AnkG intensity along axons (n ≥ 57 per group; B2) and summary scatter plot (n = 7–68; B3) showing that proteasome inhibition and Ecm29 loss significantly shorten the AIS start position relative to that seen in wild-type neurons (±SEM; *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant, one-way ANOVA followed by Dunnett’s post hoc test). Note that AIS integrity, as reflected by the peak AnkG intensity (upper panel, B2), is comparable in wild-type and Ecm29 KO neurons. (B4) Histogram ( n = 7–68 per group; ****, P < 0.0001; ns, not significant, one-way ANOVA followed by Dunnett’s post hoc test) showing effects of proteasome and NKCC1 inhibition on the changes in the AIS start position and length. (C) Similar to A, except that 7-DIV cortical neurons were costained with antibodies against pan-voltage-gated Na + channels (Pan-Na V ; C1–C3). Scale bar, 20 µm. Note that both pan-Na V and AnkG start positions in Ecm29 KO neurons exhibit an accelerated proximal shift in AIS position (C2 and C4). Data represent mean ± SEM, n > 60 cells per group from three independent experiments. ****, P < 0.0001; ns, not significant by one-way ANOVA followed by Dunnett’s post hoc test. (D) Schematic illustrating proposed AIS-associated, Ecm29 −/− proteasome-dependent modulation of GABA-evoked excitability properties in maturing neurons, requiring developmental NKCC1 down-regulation and proper subcellular localization of proteasomes.

Article Snippet: AnkG siRNA constructs (Accell SMARTpool Ank3 siRNAs), a mixture of four siRNAs (5′-CUG​GCU​UCC​UUU​ACG​UUU​U-3′, 5′-CUA​UGA​AUU​GUA​GAA​GUA​U-3′, 5′-CGC​UUA​UUU​AAA​UGU​CUU​A-3′, and 5′-UCA​UAA​UUA​AGG​AUU​UGU​A-3′), proteasome subunit Rpt1 siRNA constructs (5′-UUG​UUA​ACC​UUG​GAA​UCG​A-3′, 5′-GUC​GUA​UGC​CAA​AUU​UAG​U-3′, 5′-GCA​AGA​UGU​ACC​AAG​AUA​A-3′, and 5′-GCG​GAG​AUU​AGA​AGC​GUU​U-3′), and a scrambled siRNA (Acell Non-targeting siRNA #1: 5′-UGG​UUU​ACA​UGU​CGA​CUA​A-3′) were purchased from Dharmacon.

Techniques: Inhibition, Activity Assay, Expressing, Immunostaining

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: Cultured hippocampal neurons from WT mice were infected with p18 shRNA (mouse) lentivirus (sc-108727-V, Santa Cruz Biotechnology) or scrambled shRNA lentivirus (sc-108080, Santa Cruz Biotechnology), and co-transfected with Accell Ube3a siRNA (GE Dharmacon) or Accell Non-targeting siRNA (GE Dharmacon) at DIV 4, and 24 hr after infection, two-thirds of the medium was replaced with fresh medium.

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: Cultured hippocampal neurons from WT mice were infected with p18 shRNA (mouse) lentivirus (sc-108727-V, Santa Cruz Biotechnology) or scrambled shRNA lentivirus (sc-108080, Santa Cruz Biotechnology), and co-transfected with Accell Ube3a siRNA (GE Dharmacon) or Accell Non-targeting siRNA (GE Dharmacon) at DIV 4, and 24 hr after infection, two-thirds of the medium was replaced with fresh medium.

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 ) Images of cultured hippocampal neurons co-immunostained for lysosomal protein LAMP2 (green) and p18 (red). Insets are enlarged images of LAMP2- and p18-immunoreactive puncta along the dendrites. Arrowheads indicate co-localized puncta. Scale bar: top, 20 μm; inset, 10 μm. ( B ) Left, p18 forms a complex with p14 and MP1 in hippocampal neurons. Lysates from cultured hippocampal neurons were immunoprecipitated with an anti-p18 antibody or control IgG and probed with the indicated antibodies. Right, RagA co-immunoprecipitates RagB, RagC, p18, and p14. Lysates from mouse hippocampi were immunoprecipitated with an anti-RagA antibody or control IgG and probed with the indicated antibodies. ( C ) Images of cultured hippocampal neurons co-immunostained for p18 (magenta) and LAMP2 (red). Neurons were infected with shRNA AAV directed against p18 with GFP co-expression or scrambled shRNA control before processing for immunofluorescence assay and imaging. Scale bar, 20 μm. ( D ) Images of hippocampal neurons stained for LAMTOR4 (red). Cells were infected and processed as in ( C ). Scale bar, 10 μm; inset, 5 μm. ( E ) Images of hippocampal neurons stained for RagA (red). Cells were infected and processed as in ( C ). Scale bar, 10 μm; inset, 5 μm. ( F ) Quantification of fluorescent signals for p18 (n = 13, p<0.001), LAMTOR4 (n = 11, p<0.001), RagA (n = 6, p=0.002), and LAMP2 (n = 6, p=0.356) in control shRNA and p18 shRNA-infected neurons shown in C– E. Student’s t-test. Note that n refers to the number of culture dishes analyzed. ( G ) Left, Western blot analysis of p18, p14, LAMTOR4, RagA, RagB, and LAMP2 in enriched lysosomal fractions prepared from WT neurons transfected with Accell control or p18 siRNA. Right, quantitative analysis of blots. Results are expressed as % of values in control siRNA-transfected WT neurons and shown as means ± SEM N = 3 independent experiments, p<0.001 for p18, p14, LAMTOR4, and RagA, p=0.001 for RagB (unpaired, two-tailed Student's t-test). See also and . 10.7554/eLife.37993.007 Figure 2—source data 1. Quantitative analyses of images and Western blots used for and .

Article Snippet: Cultured hippocampal neurons from WT mice were infected with p18 shRNA (mouse) lentivirus (sc-108727-V, Santa Cruz Biotechnology) or scrambled shRNA lentivirus (sc-108080, Santa Cruz Biotechnology), and co-transfected with Accell Ube3a siRNA (GE Dharmacon) or Accell Non-targeting siRNA (GE Dharmacon) at DIV 4, and 24 hr after infection, two-thirds of the medium was replaced with fresh medium.

Techniques: Cell Culture, Immunoprecipitation, Control, Infection, shRNA, Expressing, Immunofluorescence, Imaging, Staining, Western Blot, Transfection, Two Tailed Test

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: Cultured hippocampal neurons from WT mice were infected with p18 shRNA (mouse) lentivirus (sc-108727-V, Santa Cruz Biotechnology) or scrambled shRNA lentivirus (sc-108080, Santa Cruz Biotechnology), and co-transfected with Accell Ube3a siRNA (GE Dharmacon) or Accell Non-targeting siRNA (GE Dharmacon) at DIV 4, and 24 hr after infection, two-thirds of the medium was replaced with fresh medium.

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 ) Upper panel: Representative images of Western blots labeled with p-AKT and AKT. Protein lysates from cultured hippocampal neurons transfected with the indicated constructs were prepared for Western blot analysis. Lower panel: quantitative analysis of blots. N = 3 independent experiments. This figure is paired with . ( B ) Representative images of p18 (red) and GFP in WT and AS hippocampal neurons co-infected with copGFP lentivirus and p18 shRNA or scrambled shRNA lentivirus. Scale bar, 30 µm.

Article Snippet: Cultured hippocampal neurons from WT mice were infected with p18 shRNA (mouse) lentivirus (sc-108727-V, Santa Cruz Biotechnology) or scrambled shRNA lentivirus (sc-108080, Santa Cruz Biotechnology), and co-transfected with Accell Ube3a siRNA (GE Dharmacon) or Accell Non-targeting siRNA (GE Dharmacon) at DIV 4, and 24 hr after infection, two-thirds of the medium was replaced with fresh medium.

Techniques: Western Blot, Labeling, 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 ) Representative images of dendrites of CA1 pyramidal neurons stained with anti-p18 (red) and -PSD95 (green) antibodies. Arrowheads indicate co-localized puncta. Scale bar = 10 µm. ( B ) Representative images of CA1 pyramidal neurons stained with anti-p18 (red) and -GFP (green) antibodies. Scale bar = 20 µm. ( C ) Quantitative analysis of the mean fluorescence intensity (MFI) of p18-immunoreactive puncta in GFP-positive CA1 pyramidal neurons. N = 6 mice, p<0.001, WT-siScrambled vs. WT-siP18; p<0.001, WT-siScrambled vs. AS-siScrambled; p<0.001, AS-siScrambled vs. AS-siP18; p=0.043, WT-siP18 vs. AS-siP18, two-way ANOVA with Tukey’s post-test. ( D, E ) Effects of AAV siRNA-mediated p18 KD on LTP in WT and AS mice. ( D ) Slopes of fEPSPs were normalized to the average values recorded during the 10 min baseline. ( E ) Means ± SEM of fEPSPs measured 40 min after TBS in different groups. N = 7–14 slices from four to eight mice, p=0.005, WT-siScrambled vs. WT-siP18, p<0.001, WT-siScrambled vs. AS-siScrambled, p=0.001, AS-siScrambled vs. AS-siP18, p=0.305, WT- siScrambled vs. AS-siP18, two-way ANOVA with Tukey’s post-test. Inset shows representative traces of evoked fEPSPs before and 40 min after TBS. Scale bar 0.5 mV/10 ms. See also and and . 10.7554/eLife.37993.021 Figure 6—source data 1. Source data for and .

Article Snippet: Cultured hippocampal neurons from WT mice were infected with p18 shRNA (mouse) lentivirus (sc-108727-V, Santa Cruz Biotechnology) or scrambled shRNA lentivirus (sc-108080, Santa Cruz Biotechnology), and co-transfected with Accell Ube3a siRNA (GE Dharmacon) or Accell Non-targeting siRNA (GE Dharmacon) at DIV 4, and 24 hr after infection, two-thirds of the medium was replaced with fresh medium.

Techniques: Staining, Fluorescence

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: Cultured hippocampal neurons from WT mice were infected with p18 shRNA (mouse) lentivirus (sc-108727-V, Santa Cruz Biotechnology) or scrambled shRNA lentivirus (sc-108080, Santa Cruz Biotechnology), and co-transfected with Accell Ube3a siRNA (GE Dharmacon) or Accell Non-targeting siRNA (GE Dharmacon) at DIV 4, and 24 hr after infection, two-thirds of the medium was replaced with fresh medium.

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:

Article Snippet: Cultured hippocampal neurons from WT mice were infected with p18 shRNA (mouse) lentivirus (sc-108727-V, Santa Cruz Biotechnology) or scrambled shRNA lentivirus (sc-108080, Santa Cruz Biotechnology), and co-transfected with Accell Ube3a siRNA (GE Dharmacon) or Accell Non-targeting siRNA (GE Dharmacon) at DIV 4, and 24 hr after infection, two-thirds of the medium was replaced with fresh medium.

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