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glua1 phospho serine 845 rabbit antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc glua1 phospho serine 845 rabbit antibody
    Parkin mutation/loss-of-function decreases cell-surface <t>GluA1</t> levels. a Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C, or shParkin-G430D constructs. Scale bar, 10 μm. b Quantification of cell-surface GluA1 intensity expressed as a fraction of shParkin-WT ( n ≥ 70 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 4 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM). c Representative images of surface GluA1 staining (red) in 14–16 DIV Parkin KO hippocampal neurons expressing shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C, or shParkin-G430D constructs and non-transduced KO control. Scale bar, 10 μm. d Quantification of cell-surface GluA1 intensity expressed as a fraction of Parkin KO ( n ≥ 50 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM)
    Glua1 Phospho Serine 845 Rabbit Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glua1 phospho serine 845 rabbit antibody/product/Cell Signaling Technology Inc
    Average 95 stars, based on 133 article reviews
    glua1 phospho serine 845 rabbit antibody - by Bioz Stars, 2026-03
    95/100 stars

    Images

    1) Product Images from "Parkinson’s disease-linked Parkin mutations impair glutamatergic signaling in hippocampal neurons"

    Article Title: Parkinson’s disease-linked Parkin mutations impair glutamatergic signaling in hippocampal neurons

    Journal: BMC Biology

    doi: 10.1186/s12915-018-0567-7

    Parkin mutation/loss-of-function decreases cell-surface GluA1 levels. a Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C, or shParkin-G430D constructs. Scale bar, 10 μm. b Quantification of cell-surface GluA1 intensity expressed as a fraction of shParkin-WT ( n ≥ 70 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 4 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM). c Representative images of surface GluA1 staining (red) in 14–16 DIV Parkin KO hippocampal neurons expressing shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C, or shParkin-G430D constructs and non-transduced KO control. Scale bar, 10 μm. d Quantification of cell-surface GluA1 intensity expressed as a fraction of Parkin KO ( n ≥ 50 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM)
    Figure Legend Snippet: Parkin mutation/loss-of-function decreases cell-surface GluA1 levels. a Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C, or shParkin-G430D constructs. Scale bar, 10 μm. b Quantification of cell-surface GluA1 intensity expressed as a fraction of shParkin-WT ( n ≥ 70 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 4 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM). c Representative images of surface GluA1 staining (red) in 14–16 DIV Parkin KO hippocampal neurons expressing shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C, or shParkin-G430D constructs and non-transduced KO control. Scale bar, 10 μm. d Quantification of cell-surface GluA1 intensity expressed as a fraction of Parkin KO ( n ≥ 50 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM)

    Techniques Used: Mutagenesis, Staining, Expressing, Construct, Control

    Parkin mutation/loss-of-function decreases cell-surface GluN1 levels. a Representative images of surface GluN1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C, or shParkin-G430D constructs. Scale bar, 10 μm. b Quantification of cell-surface GluN1 intensity expressed as a fraction of shParkin-WT ( n ≥ 50 fields of view per condition with > 100 GluN1 puncta per field, results confirmed in 4 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM). Scale bar, 10 μm. c Representative images of surface GluN1 staining (red) in 14–16 DIV Parkin KO hippocampal neurons expressing shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C or shParkin-G430D constructs, and non-transduced KO control. Scale bar, 10 μm. d Quantification of cell-surface GluN1 intensity expressed as a fraction of Parkin KO ( n ≥ 50 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM)
    Figure Legend Snippet: Parkin mutation/loss-of-function decreases cell-surface GluN1 levels. a Representative images of surface GluN1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C, or shParkin-G430D constructs. Scale bar, 10 μm. b Quantification of cell-surface GluN1 intensity expressed as a fraction of shParkin-WT ( n ≥ 50 fields of view per condition with > 100 GluN1 puncta per field, results confirmed in 4 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM). Scale bar, 10 μm. c Representative images of surface GluN1 staining (red) in 14–16 DIV Parkin KO hippocampal neurons expressing shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C or shParkin-G430D constructs, and non-transduced KO control. Scale bar, 10 μm. d Quantification of cell-surface GluN1 intensity expressed as a fraction of Parkin KO ( n ≥ 50 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM)

    Techniques Used: Mutagenesis, Staining, Expressing, Construct, Control

    Parkin E3 ligase activity is required to maintain cell-surface NMDARs but not AMPARs. a Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing shParkin +/− WT, C431S, or W403A Parkin constructs. Scale bar, 10 μm. b Same condition as ( a ), but for surface GluN1 staining (red). Scale bar, 10 μm. c Quantification of cell- surface GluA1 intensity expressed as a fraction of shParkin control ( n ≥ 40 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM). d Quantification of cell-surface GluN1 intensity expressed as a fraction of shParkin control ( n ≥ 40 fields of view per condition with > 100 GluN1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM)
    Figure Legend Snippet: Parkin E3 ligase activity is required to maintain cell-surface NMDARs but not AMPARs. a Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing shParkin +/− WT, C431S, or W403A Parkin constructs. Scale bar, 10 μm. b Same condition as ( a ), but for surface GluN1 staining (red). Scale bar, 10 μm. c Quantification of cell- surface GluA1 intensity expressed as a fraction of shParkin control ( n ≥ 40 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM). d Quantification of cell-surface GluN1 intensity expressed as a fraction of shParkin control ( n ≥ 40 fields of view per condition with > 100 GluN1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM)

    Techniques Used: Activity Assay, Staining, Expressing, Construct, Control

    Parkin-mediated GluN1 ubiquitination is impaired in pathogenic mutants. a Representative immunoblots for GFP immunoprecipitation (IP) from HEK293T cell lysates expressing Myc/Myc-Parkin, GFP-GluA1/-GluA2/-GluN1/-GluN2B, and HA-ubiquitin, probed for HA and GFP. Ubiquitin immunoreactivity used for quantification is marked on HA blots. b Quantification of GFP-GluA or GluN ubiquitination, expressed as the ratio of marked HA blot intensity ( a ) with Myc-Parkin (+) to Myc control (−), then normalized to immunoprecipitated GFP, GFP-GluA or GluN ( n = 3 experiments, * P < 0.05; one-way ANOVA, error bars represent SEM). c Representative Myc and GFP immunoblots for Myc IP from HEK293T cell lysates expressing Myc-Parkin and GFP-GluA1/-GluA2/-GluN1/-GluN2B. Arrowhead indicates immunoprecipitated Myc-Parkin (just below IgG band). d Representative HA and Flag immunoblots for Flag IP from HEK293T cell lysates expressing Flag-GluN1, GFP control/GFP-Parkin WT/C431S/W403A, and HA-ubiquitin. Arrowhead indicates immunoprecipitated Flag-GluN1. Ubiquitin immunoreactivity used for quantification is marked on HA blots. e Quantification of Flag-GluN1 ubiquitination by measurement of marked HA blot intensity ( d ), normalized to immunoprecipitated Flag-GluN1 and reported as a fraction of GFP control ( n = 3 experiments, ** P < 0.01, *** P < 0.001, one-way ANOVA, error bars represent SEM). f Representative HA and Flag immunoblots for Flag IP from HEK293T cell lysates expressing Flag-GluN1, GFP control/GFP-Parkin WT/T240M/R275W/R334C/G430D constructs, and HA-ubiquitin. Arrowhead indicates immunoprecipitated Flag-GluN1. Ubiquitin immunoreactivity used for quantification is marked on HA blots. g Quantification of Flag-GluN1 ubiquitination by measurement of marked HA intensity ( f ), normalized to immunoprecipitated Flag-GluN1 and reported as a fraction of GFP condition ( n = 3 experiments, * P < 0.05; ** P < 0.01, *** P < 0.001, one-way ANOVA, error bars represent SEM)
    Figure Legend Snippet: Parkin-mediated GluN1 ubiquitination is impaired in pathogenic mutants. a Representative immunoblots for GFP immunoprecipitation (IP) from HEK293T cell lysates expressing Myc/Myc-Parkin, GFP-GluA1/-GluA2/-GluN1/-GluN2B, and HA-ubiquitin, probed for HA and GFP. Ubiquitin immunoreactivity used for quantification is marked on HA blots. b Quantification of GFP-GluA or GluN ubiquitination, expressed as the ratio of marked HA blot intensity ( a ) with Myc-Parkin (+) to Myc control (−), then normalized to immunoprecipitated GFP, GFP-GluA or GluN ( n = 3 experiments, * P < 0.05; one-way ANOVA, error bars represent SEM). c Representative Myc and GFP immunoblots for Myc IP from HEK293T cell lysates expressing Myc-Parkin and GFP-GluA1/-GluA2/-GluN1/-GluN2B. Arrowhead indicates immunoprecipitated Myc-Parkin (just below IgG band). d Representative HA and Flag immunoblots for Flag IP from HEK293T cell lysates expressing Flag-GluN1, GFP control/GFP-Parkin WT/C431S/W403A, and HA-ubiquitin. Arrowhead indicates immunoprecipitated Flag-GluN1. Ubiquitin immunoreactivity used for quantification is marked on HA blots. e Quantification of Flag-GluN1 ubiquitination by measurement of marked HA blot intensity ( d ), normalized to immunoprecipitated Flag-GluN1 and reported as a fraction of GFP control ( n = 3 experiments, ** P < 0.01, *** P < 0.001, one-way ANOVA, error bars represent SEM). f Representative HA and Flag immunoblots for Flag IP from HEK293T cell lysates expressing Flag-GluN1, GFP control/GFP-Parkin WT/T240M/R275W/R334C/G430D constructs, and HA-ubiquitin. Arrowhead indicates immunoprecipitated Flag-GluN1. Ubiquitin immunoreactivity used for quantification is marked on HA blots. g Quantification of Flag-GluN1 ubiquitination by measurement of marked HA intensity ( f ), normalized to immunoprecipitated Flag-GluN1 and reported as a fraction of GFP condition ( n = 3 experiments, * P < 0.05; ** P < 0.01, *** P < 0.001, one-way ANOVA, error bars represent SEM)

    Techniques Used: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Expressing, Control, Construct

    Parkin mutation/knockdown impairs the induction of LTD. a Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin or shParkin-WT, under the control condition (no treatment) or after chemical LTP (cLTP) induction. Scale bar, 10 μm. b Quantification of the ratio of GluA1 intensity after cLTP induction to the control condition for neurons expressing GFP, shParkin, or shParkin-WT. c Same as ( a ), but for control condition or chemical LTD (cLTD) induction. Scale bar, 10 μm. d Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing GFP, shParkin, or shParkin-WT. e Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs, under the control condition or after cLTD induction. Scale bar, 10 μm. f Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing GFP, shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs. For panels ( b ) and ( d ), n ≥ 50 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, unpaired t test. For panel ( f ), n ≥ 40 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM
    Figure Legend Snippet: Parkin mutation/knockdown impairs the induction of LTD. a Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin or shParkin-WT, under the control condition (no treatment) or after chemical LTP (cLTP) induction. Scale bar, 10 μm. b Quantification of the ratio of GluA1 intensity after cLTP induction to the control condition for neurons expressing GFP, shParkin, or shParkin-WT. c Same as ( a ), but for control condition or chemical LTD (cLTD) induction. Scale bar, 10 μm. d Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing GFP, shParkin, or shParkin-WT. e Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs, under the control condition or after cLTD induction. Scale bar, 10 μm. f Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing GFP, shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs. For panels ( b ) and ( d ), n ≥ 50 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, unpaired t test. For panel ( f ), n ≥ 40 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM

    Techniques Used: Mutagenesis, Knockdown, Staining, Expressing, Control, Construct

    Parkin mutation/knockout impairs the induction of LTD but not LTP. a Representative images of surface GluA1 staining (red) in 14–16 DIV Parkin KO hippocampal neurons expressing shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs, and non-transduced KO control under the control condition (no treatment) or after chemical LTP (cLTP) induction. Scale bar, 10 μm. b Quantification of the ratio of GluA1 intensity after cLTP induction to the control condition for neurons expressing the above Parkin constructs. c Same as ( a ), but for control condition or chemical LTD (cLTD) induction. Scale bar, 10 μm. d Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing above Parkin constructs. For panels ( b ) and ( d ), n ≥ 40 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM
    Figure Legend Snippet: Parkin mutation/knockout impairs the induction of LTD but not LTP. a Representative images of surface GluA1 staining (red) in 14–16 DIV Parkin KO hippocampal neurons expressing shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs, and non-transduced KO control under the control condition (no treatment) or after chemical LTP (cLTP) induction. Scale bar, 10 μm. b Quantification of the ratio of GluA1 intensity after cLTP induction to the control condition for neurons expressing the above Parkin constructs. c Same as ( a ), but for control condition or chemical LTD (cLTD) induction. Scale bar, 10 μm. d Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing above Parkin constructs. For panels ( b ) and ( d ), n ≥ 40 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM

    Techniques Used: Mutagenesis, Knock-Out, Staining, Expressing, Construct, Control



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    Image Search Results


    Parkin mutation/loss-of-function decreases cell-surface GluA1 levels. a Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C, or shParkin-G430D constructs. Scale bar, 10 μm. b Quantification of cell-surface GluA1 intensity expressed as a fraction of shParkin-WT ( n ≥ 70 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 4 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM). c Representative images of surface GluA1 staining (red) in 14–16 DIV Parkin KO hippocampal neurons expressing shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C, or shParkin-G430D constructs and non-transduced KO control. Scale bar, 10 μm. d Quantification of cell-surface GluA1 intensity expressed as a fraction of Parkin KO ( n ≥ 50 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM)

    Journal: BMC Biology

    Article Title: Parkinson’s disease-linked Parkin mutations impair glutamatergic signaling in hippocampal neurons

    doi: 10.1186/s12915-018-0567-7

    Figure Lengend Snippet: Parkin mutation/loss-of-function decreases cell-surface GluA1 levels. a Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C, or shParkin-G430D constructs. Scale bar, 10 μm. b Quantification of cell-surface GluA1 intensity expressed as a fraction of shParkin-WT ( n ≥ 70 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 4 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM). c Representative images of surface GluA1 staining (red) in 14–16 DIV Parkin KO hippocampal neurons expressing shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C, or shParkin-G430D constructs and non-transduced KO control. Scale bar, 10 μm. d Quantification of cell-surface GluA1 intensity expressed as a fraction of Parkin KO ( n ≥ 50 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM)

    Article Snippet: The following primary antibodies and dilutions were used for western blot and immunoprecipitation: mouse Parkin (Prk8, 1:1000; Santa Cruz Biotechnology), GluN1 (1:500; EMD Millipore), GluN2A (1:500; EMD Millipore), GluN2B (1:500; Neuromab), GluA1 phospho-Serine 845 rabbit antibody (1:1,000; Cell Signaling Technology), mouse tubulin (1:10,000; Sigma), rabbit tubulin (1:10,000; Abcam), rabbit GFP (1:1000; Invitrogen), mouse Myc (1:500; Santa Cruz Biotechnology), mouse HA (1:500; Santa Cruz Biotechnology), and mouse Flag M2 (1:5,000; Sigma).

    Techniques: Mutagenesis, Staining, Expressing, Construct, Control

    Parkin mutation/loss-of-function decreases cell-surface GluN1 levels. a Representative images of surface GluN1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C, or shParkin-G430D constructs. Scale bar, 10 μm. b Quantification of cell-surface GluN1 intensity expressed as a fraction of shParkin-WT ( n ≥ 50 fields of view per condition with > 100 GluN1 puncta per field, results confirmed in 4 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM). Scale bar, 10 μm. c Representative images of surface GluN1 staining (red) in 14–16 DIV Parkin KO hippocampal neurons expressing shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C or shParkin-G430D constructs, and non-transduced KO control. Scale bar, 10 μm. d Quantification of cell-surface GluN1 intensity expressed as a fraction of Parkin KO ( n ≥ 50 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM)

    Journal: BMC Biology

    Article Title: Parkinson’s disease-linked Parkin mutations impair glutamatergic signaling in hippocampal neurons

    doi: 10.1186/s12915-018-0567-7

    Figure Lengend Snippet: Parkin mutation/loss-of-function decreases cell-surface GluN1 levels. a Representative images of surface GluN1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C, or shParkin-G430D constructs. Scale bar, 10 μm. b Quantification of cell-surface GluN1 intensity expressed as a fraction of shParkin-WT ( n ≥ 50 fields of view per condition with > 100 GluN1 puncta per field, results confirmed in 4 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM). Scale bar, 10 μm. c Representative images of surface GluN1 staining (red) in 14–16 DIV Parkin KO hippocampal neurons expressing shParkin, shParkin-WT, shParkin-T240M, shParkin-R275W, shParkin-R334C or shParkin-G430D constructs, and non-transduced KO control. Scale bar, 10 μm. d Quantification of cell-surface GluN1 intensity expressed as a fraction of Parkin KO ( n ≥ 50 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM)

    Article Snippet: The following primary antibodies and dilutions were used for western blot and immunoprecipitation: mouse Parkin (Prk8, 1:1000; Santa Cruz Biotechnology), GluN1 (1:500; EMD Millipore), GluN2A (1:500; EMD Millipore), GluN2B (1:500; Neuromab), GluA1 phospho-Serine 845 rabbit antibody (1:1,000; Cell Signaling Technology), mouse tubulin (1:10,000; Sigma), rabbit tubulin (1:10,000; Abcam), rabbit GFP (1:1000; Invitrogen), mouse Myc (1:500; Santa Cruz Biotechnology), mouse HA (1:500; Santa Cruz Biotechnology), and mouse Flag M2 (1:5,000; Sigma).

    Techniques: Mutagenesis, Staining, Expressing, Construct, Control

    Parkin E3 ligase activity is required to maintain cell-surface NMDARs but not AMPARs. a Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing shParkin +/− WT, C431S, or W403A Parkin constructs. Scale bar, 10 μm. b Same condition as ( a ), but for surface GluN1 staining (red). Scale bar, 10 μm. c Quantification of cell- surface GluA1 intensity expressed as a fraction of shParkin control ( n ≥ 40 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM). d Quantification of cell-surface GluN1 intensity expressed as a fraction of shParkin control ( n ≥ 40 fields of view per condition with > 100 GluN1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM)

    Journal: BMC Biology

    Article Title: Parkinson’s disease-linked Parkin mutations impair glutamatergic signaling in hippocampal neurons

    doi: 10.1186/s12915-018-0567-7

    Figure Lengend Snippet: Parkin E3 ligase activity is required to maintain cell-surface NMDARs but not AMPARs. a Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing shParkin +/− WT, C431S, or W403A Parkin constructs. Scale bar, 10 μm. b Same condition as ( a ), but for surface GluN1 staining (red). Scale bar, 10 μm. c Quantification of cell- surface GluA1 intensity expressed as a fraction of shParkin control ( n ≥ 40 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM). d Quantification of cell-surface GluN1 intensity expressed as a fraction of shParkin control ( n ≥ 40 fields of view per condition with > 100 GluN1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM)

    Article Snippet: The following primary antibodies and dilutions were used for western blot and immunoprecipitation: mouse Parkin (Prk8, 1:1000; Santa Cruz Biotechnology), GluN1 (1:500; EMD Millipore), GluN2A (1:500; EMD Millipore), GluN2B (1:500; Neuromab), GluA1 phospho-Serine 845 rabbit antibody (1:1,000; Cell Signaling Technology), mouse tubulin (1:10,000; Sigma), rabbit tubulin (1:10,000; Abcam), rabbit GFP (1:1000; Invitrogen), mouse Myc (1:500; Santa Cruz Biotechnology), mouse HA (1:500; Santa Cruz Biotechnology), and mouse Flag M2 (1:5,000; Sigma).

    Techniques: Activity Assay, Staining, Expressing, Construct, Control

    Parkin-mediated GluN1 ubiquitination is impaired in pathogenic mutants. a Representative immunoblots for GFP immunoprecipitation (IP) from HEK293T cell lysates expressing Myc/Myc-Parkin, GFP-GluA1/-GluA2/-GluN1/-GluN2B, and HA-ubiquitin, probed for HA and GFP. Ubiquitin immunoreactivity used for quantification is marked on HA blots. b Quantification of GFP-GluA or GluN ubiquitination, expressed as the ratio of marked HA blot intensity ( a ) with Myc-Parkin (+) to Myc control (−), then normalized to immunoprecipitated GFP, GFP-GluA or GluN ( n = 3 experiments, * P < 0.05; one-way ANOVA, error bars represent SEM). c Representative Myc and GFP immunoblots for Myc IP from HEK293T cell lysates expressing Myc-Parkin and GFP-GluA1/-GluA2/-GluN1/-GluN2B. Arrowhead indicates immunoprecipitated Myc-Parkin (just below IgG band). d Representative HA and Flag immunoblots for Flag IP from HEK293T cell lysates expressing Flag-GluN1, GFP control/GFP-Parkin WT/C431S/W403A, and HA-ubiquitin. Arrowhead indicates immunoprecipitated Flag-GluN1. Ubiquitin immunoreactivity used for quantification is marked on HA blots. e Quantification of Flag-GluN1 ubiquitination by measurement of marked HA blot intensity ( d ), normalized to immunoprecipitated Flag-GluN1 and reported as a fraction of GFP control ( n = 3 experiments, ** P < 0.01, *** P < 0.001, one-way ANOVA, error bars represent SEM). f Representative HA and Flag immunoblots for Flag IP from HEK293T cell lysates expressing Flag-GluN1, GFP control/GFP-Parkin WT/T240M/R275W/R334C/G430D constructs, and HA-ubiquitin. Arrowhead indicates immunoprecipitated Flag-GluN1. Ubiquitin immunoreactivity used for quantification is marked on HA blots. g Quantification of Flag-GluN1 ubiquitination by measurement of marked HA intensity ( f ), normalized to immunoprecipitated Flag-GluN1 and reported as a fraction of GFP condition ( n = 3 experiments, * P < 0.05; ** P < 0.01, *** P < 0.001, one-way ANOVA, error bars represent SEM)

    Journal: BMC Biology

    Article Title: Parkinson’s disease-linked Parkin mutations impair glutamatergic signaling in hippocampal neurons

    doi: 10.1186/s12915-018-0567-7

    Figure Lengend Snippet: Parkin-mediated GluN1 ubiquitination is impaired in pathogenic mutants. a Representative immunoblots for GFP immunoprecipitation (IP) from HEK293T cell lysates expressing Myc/Myc-Parkin, GFP-GluA1/-GluA2/-GluN1/-GluN2B, and HA-ubiquitin, probed for HA and GFP. Ubiquitin immunoreactivity used for quantification is marked on HA blots. b Quantification of GFP-GluA or GluN ubiquitination, expressed as the ratio of marked HA blot intensity ( a ) with Myc-Parkin (+) to Myc control (−), then normalized to immunoprecipitated GFP, GFP-GluA or GluN ( n = 3 experiments, * P < 0.05; one-way ANOVA, error bars represent SEM). c Representative Myc and GFP immunoblots for Myc IP from HEK293T cell lysates expressing Myc-Parkin and GFP-GluA1/-GluA2/-GluN1/-GluN2B. Arrowhead indicates immunoprecipitated Myc-Parkin (just below IgG band). d Representative HA and Flag immunoblots for Flag IP from HEK293T cell lysates expressing Flag-GluN1, GFP control/GFP-Parkin WT/C431S/W403A, and HA-ubiquitin. Arrowhead indicates immunoprecipitated Flag-GluN1. Ubiquitin immunoreactivity used for quantification is marked on HA blots. e Quantification of Flag-GluN1 ubiquitination by measurement of marked HA blot intensity ( d ), normalized to immunoprecipitated Flag-GluN1 and reported as a fraction of GFP control ( n = 3 experiments, ** P < 0.01, *** P < 0.001, one-way ANOVA, error bars represent SEM). f Representative HA and Flag immunoblots for Flag IP from HEK293T cell lysates expressing Flag-GluN1, GFP control/GFP-Parkin WT/T240M/R275W/R334C/G430D constructs, and HA-ubiquitin. Arrowhead indicates immunoprecipitated Flag-GluN1. Ubiquitin immunoreactivity used for quantification is marked on HA blots. g Quantification of Flag-GluN1 ubiquitination by measurement of marked HA intensity ( f ), normalized to immunoprecipitated Flag-GluN1 and reported as a fraction of GFP condition ( n = 3 experiments, * P < 0.05; ** P < 0.01, *** P < 0.001, one-way ANOVA, error bars represent SEM)

    Article Snippet: The following primary antibodies and dilutions were used for western blot and immunoprecipitation: mouse Parkin (Prk8, 1:1000; Santa Cruz Biotechnology), GluN1 (1:500; EMD Millipore), GluN2A (1:500; EMD Millipore), GluN2B (1:500; Neuromab), GluA1 phospho-Serine 845 rabbit antibody (1:1,000; Cell Signaling Technology), mouse tubulin (1:10,000; Sigma), rabbit tubulin (1:10,000; Abcam), rabbit GFP (1:1000; Invitrogen), mouse Myc (1:500; Santa Cruz Biotechnology), mouse HA (1:500; Santa Cruz Biotechnology), and mouse Flag M2 (1:5,000; Sigma).

    Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Expressing, Control, Construct

    Parkin mutation/knockdown impairs the induction of LTD. a Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin or shParkin-WT, under the control condition (no treatment) or after chemical LTP (cLTP) induction. Scale bar, 10 μm. b Quantification of the ratio of GluA1 intensity after cLTP induction to the control condition for neurons expressing GFP, shParkin, or shParkin-WT. c Same as ( a ), but for control condition or chemical LTD (cLTD) induction. Scale bar, 10 μm. d Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing GFP, shParkin, or shParkin-WT. e Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs, under the control condition or after cLTD induction. Scale bar, 10 μm. f Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing GFP, shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs. For panels ( b ) and ( d ), n ≥ 50 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, unpaired t test. For panel ( f ), n ≥ 40 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM

    Journal: BMC Biology

    Article Title: Parkinson’s disease-linked Parkin mutations impair glutamatergic signaling in hippocampal neurons

    doi: 10.1186/s12915-018-0567-7

    Figure Lengend Snippet: Parkin mutation/knockdown impairs the induction of LTD. a Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin or shParkin-WT, under the control condition (no treatment) or after chemical LTP (cLTP) induction. Scale bar, 10 μm. b Quantification of the ratio of GluA1 intensity after cLTP induction to the control condition for neurons expressing GFP, shParkin, or shParkin-WT. c Same as ( a ), but for control condition or chemical LTD (cLTD) induction. Scale bar, 10 μm. d Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing GFP, shParkin, or shParkin-WT. e Representative images of surface GluA1 staining (red) in 14–16 DIV hippocampal neurons expressing GFP, shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs, under the control condition or after cLTD induction. Scale bar, 10 μm. f Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing GFP, shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs. For panels ( b ) and ( d ), n ≥ 50 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, unpaired t test. For panel ( f ), n ≥ 40 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 3 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM

    Article Snippet: The following primary antibodies and dilutions were used for western blot and immunoprecipitation: mouse Parkin (Prk8, 1:1000; Santa Cruz Biotechnology), GluN1 (1:500; EMD Millipore), GluN2A (1:500; EMD Millipore), GluN2B (1:500; Neuromab), GluA1 phospho-Serine 845 rabbit antibody (1:1,000; Cell Signaling Technology), mouse tubulin (1:10,000; Sigma), rabbit tubulin (1:10,000; Abcam), rabbit GFP (1:1000; Invitrogen), mouse Myc (1:500; Santa Cruz Biotechnology), mouse HA (1:500; Santa Cruz Biotechnology), and mouse Flag M2 (1:5,000; Sigma).

    Techniques: Mutagenesis, Knockdown, Staining, Expressing, Control, Construct

    Parkin mutation/knockout impairs the induction of LTD but not LTP. a Representative images of surface GluA1 staining (red) in 14–16 DIV Parkin KO hippocampal neurons expressing shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs, and non-transduced KO control under the control condition (no treatment) or after chemical LTP (cLTP) induction. Scale bar, 10 μm. b Quantification of the ratio of GluA1 intensity after cLTP induction to the control condition for neurons expressing the above Parkin constructs. c Same as ( a ), but for control condition or chemical LTD (cLTD) induction. Scale bar, 10 μm. d Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing above Parkin constructs. For panels ( b ) and ( d ), n ≥ 40 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM

    Journal: BMC Biology

    Article Title: Parkinson’s disease-linked Parkin mutations impair glutamatergic signaling in hippocampal neurons

    doi: 10.1186/s12915-018-0567-7

    Figure Lengend Snippet: Parkin mutation/knockout impairs the induction of LTD but not LTP. a Representative images of surface GluA1 staining (red) in 14–16 DIV Parkin KO hippocampal neurons expressing shParkin or shParkin-WT/-T240M/-R275W/-R334C/-G430D constructs, and non-transduced KO control under the control condition (no treatment) or after chemical LTP (cLTP) induction. Scale bar, 10 μm. b Quantification of the ratio of GluA1 intensity after cLTP induction to the control condition for neurons expressing the above Parkin constructs. c Same as ( a ), but for control condition or chemical LTD (cLTD) induction. Scale bar, 10 μm. d Quantification of the ratio of GluA1 intensity after cLTD induction to the control condition for neurons expressing above Parkin constructs. For panels ( b ) and ( d ), n ≥ 40 fields of view per condition with > 100 GluA1 puncta per field, results confirmed in 2 independent experiments. *** P < 0.001, one-way ANOVA, error bars represent SEM

    Article Snippet: The following primary antibodies and dilutions were used for western blot and immunoprecipitation: mouse Parkin (Prk8, 1:1000; Santa Cruz Biotechnology), GluN1 (1:500; EMD Millipore), GluN2A (1:500; EMD Millipore), GluN2B (1:500; Neuromab), GluA1 phospho-Serine 845 rabbit antibody (1:1,000; Cell Signaling Technology), mouse tubulin (1:10,000; Sigma), rabbit tubulin (1:10,000; Abcam), rabbit GFP (1:1000; Invitrogen), mouse Myc (1:500; Santa Cruz Biotechnology), mouse HA (1:500; Santa Cruz Biotechnology), and mouse Flag M2 (1:5,000; Sigma).

    Techniques: Mutagenesis, Knock-Out, Staining, Expressing, Construct, Control