agp  (Alomone Labs)


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    Alomone Labs agp
    Agp, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    agp  (Alomone Labs)


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    Alomone Labs agp
    Agp, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    guinea pig anti ampa receptor 2 subunit  (Alomone Labs)


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    Alomone Labs guinea pig anti ampa receptor 2 subunit
    Validation of differentially expressed genes using qRT-PCR. ( A–E ) qPCR verification of the expression of genes involved in biological process identified as enriched by GO analysis compared with WT control. (F) Fold change expression of Ca 2+ -permeable AMPAR subunit Gria1 , Gria3 and Gria4 mRNAs, relative to WT motor neurons at E12.5. (G) Relative expression of Adarb1 mRNA in SOD1 G93A motor neurons at E12.5. ( H ) Schema showing the position of the fully complementary miR-124 target site at the 5′-end of the mouse <t>Gria2</t> , 3′-UTR. The seed region of miR-124 is shown. Data represent mean ± SEM, unpaired student t -test, n = 5–7 biological replicates, * P < 0.05.
    Guinea Pig Anti Ampa Receptor 2 Subunit, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    1) Product Images from "α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis"

    Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

    Journal: Brain Communications

    doi: 10.1093/braincomms/fcac081

    Validation of differentially expressed genes using qRT-PCR. ( A–E ) qPCR verification of the expression of genes involved in biological process identified as enriched by GO analysis compared with WT control. (F) Fold change expression of Ca 2+ -permeable AMPAR subunit Gria1 , Gria3 and Gria4 mRNAs, relative to WT motor neurons at E12.5. (G) Relative expression of Adarb1 mRNA in SOD1 G93A motor neurons at E12.5. ( H ) Schema showing the position of the fully complementary miR-124 target site at the 5′-end of the mouse Gria2 , 3′-UTR. The seed region of miR-124 is shown. Data represent mean ± SEM, unpaired student t -test, n = 5–7 biological replicates, * P < 0.05.
    Figure Legend Snippet: Validation of differentially expressed genes using qRT-PCR. ( A–E ) qPCR verification of the expression of genes involved in biological process identified as enriched by GO analysis compared with WT control. (F) Fold change expression of Ca 2+ -permeable AMPAR subunit Gria1 , Gria3 and Gria4 mRNAs, relative to WT motor neurons at E12.5. (G) Relative expression of Adarb1 mRNA in SOD1 G93A motor neurons at E12.5. ( H ) Schema showing the position of the fully complementary miR-124 target site at the 5′-end of the mouse Gria2 , 3′-UTR. The seed region of miR-124 is shown. Data represent mean ± SEM, unpaired student t -test, n = 5–7 biological replicates, * P < 0.05.

    Techniques Used: Quantitative RT-PCR, Expressing

    Expression of GluA2 in spinal cords of embryonic SOD1 G93A mice. Cross-sections of lumbar spinal cord from WT (HB9:GFP; WT) and SOD1 G93A (SOD1 G93A ; HB9:GFP) mice at (A–J) E12.5 and ( K–T ) E17.5. Double-immunolabelling for GFP, GluA2 and NeuN (Neuronal nuclei). Plots represent quantification analysis of GluA2 signal intensity in HB9:GFP motor neurons at ( U ) E12.5 and ( V ) E17.5. Data represent mean ± SEM, unpaired student t -test performed on n = 4 biological replicates, ∼50 neurons analysed per biological replicate, * P < 0.05. Scale bars 50 μm.
    Figure Legend Snippet: Expression of GluA2 in spinal cords of embryonic SOD1 G93A mice. Cross-sections of lumbar spinal cord from WT (HB9:GFP; WT) and SOD1 G93A (SOD1 G93A ; HB9:GFP) mice at (A–J) E12.5 and ( K–T ) E17.5. Double-immunolabelling for GFP, GluA2 and NeuN (Neuronal nuclei). Plots represent quantification analysis of GluA2 signal intensity in HB9:GFP motor neurons at ( U ) E12.5 and ( V ) E17.5. Data represent mean ± SEM, unpaired student t -test performed on n = 4 biological replicates, ∼50 neurons analysed per biological replicate, * P < 0.05. Scale bars 50 μm.

    Techniques Used: Expressing

    Expression of GRIA2 and ADAR2 in iPSC motor neurons derived from ALS patients with SOD1 mutations and healthy control lines. Representative images of iPSC mature motor neurons derived from ( A–E ) healthy control line and ( F–J ) SOD1 I114T line, immunolabelled with ChAT, GluA2 and TUJ1, counterstained with Hoechst. ( K ) Plot represents quantification analysis of GluA2 signal intensity in iPSC motor neurons. Data represent mean ± SEM, unpaired student t -test performed on n = 3 biological replicates, 50 neurons analysed per biological replicate. (L) Fold change expression of GRIA2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. ( M ) Fold change expression of ADAR2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. Data represent mean ± SEM, n = 3 biological replicates, one-way ANOVA with Dunnett's multiple comparison test, * P < 0.01, ** P < 0.005. Scale bars 50 μm.
    Figure Legend Snippet: Expression of GRIA2 and ADAR2 in iPSC motor neurons derived from ALS patients with SOD1 mutations and healthy control lines. Representative images of iPSC mature motor neurons derived from ( A–E ) healthy control line and ( F–J ) SOD1 I114T line, immunolabelled with ChAT, GluA2 and TUJ1, counterstained with Hoechst. ( K ) Plot represents quantification analysis of GluA2 signal intensity in iPSC motor neurons. Data represent mean ± SEM, unpaired student t -test performed on n = 3 biological replicates, 50 neurons analysed per biological replicate. (L) Fold change expression of GRIA2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. ( M ) Fold change expression of ADAR2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. Data represent mean ± SEM, n = 3 biological replicates, one-way ANOVA with Dunnett's multiple comparison test, * P < 0.01, ** P < 0.005. Scale bars 50 μm.

    Techniques Used: Expressing, Derivative Assay, Quantitative RT-PCR

    rabbit anti glua2  (Alomone Labs)


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    Alomone Labs rabbit anti glua2
    Validation of differentially expressed genes using qRT-PCR. ( A–E ) qPCR verification of the expression of genes involved in biological process identified as enriched by GO analysis compared with WT control. (F) Fold change expression of Ca 2+ -permeable AMPAR subunit Gria1 , Gria3 and Gria4 mRNAs, relative to WT motor neurons at E12.5. (G) Relative expression of Adarb1 mRNA in SOD1 G93A motor neurons at E12.5. ( H ) Schema showing the position of the fully complementary miR-124 target site at the 5′-end of the mouse <t>Gria2</t> , 3′-UTR. The seed region of miR-124 is shown. Data represent mean ± SEM, unpaired student t -test, n = 5–7 biological replicates, * P < 0.05.
    Rabbit Anti Glua2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    rabbit anti glua2 - by Bioz Stars, 2023-02
    94/100 stars

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    1) Product Images from "α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis"

    Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

    Journal: Brain Communications

    doi: 10.1093/braincomms/fcac081

    Validation of differentially expressed genes using qRT-PCR. ( A–E ) qPCR verification of the expression of genes involved in biological process identified as enriched by GO analysis compared with WT control. (F) Fold change expression of Ca 2+ -permeable AMPAR subunit Gria1 , Gria3 and Gria4 mRNAs, relative to WT motor neurons at E12.5. (G) Relative expression of Adarb1 mRNA in SOD1 G93A motor neurons at E12.5. ( H ) Schema showing the position of the fully complementary miR-124 target site at the 5′-end of the mouse Gria2 , 3′-UTR. The seed region of miR-124 is shown. Data represent mean ± SEM, unpaired student t -test, n = 5–7 biological replicates, * P < 0.05.
    Figure Legend Snippet: Validation of differentially expressed genes using qRT-PCR. ( A–E ) qPCR verification of the expression of genes involved in biological process identified as enriched by GO analysis compared with WT control. (F) Fold change expression of Ca 2+ -permeable AMPAR subunit Gria1 , Gria3 and Gria4 mRNAs, relative to WT motor neurons at E12.5. (G) Relative expression of Adarb1 mRNA in SOD1 G93A motor neurons at E12.5. ( H ) Schema showing the position of the fully complementary miR-124 target site at the 5′-end of the mouse Gria2 , 3′-UTR. The seed region of miR-124 is shown. Data represent mean ± SEM, unpaired student t -test, n = 5–7 biological replicates, * P < 0.05.

    Techniques Used: Quantitative RT-PCR, Expressing

    Expression of GluA2 in spinal cords of embryonic SOD1 G93A mice. Cross-sections of lumbar spinal cord from WT (HB9:GFP; WT) and SOD1 G93A (SOD1 G93A ; HB9:GFP) mice at (A–J) E12.5 and ( K–T ) E17.5. Double-immunolabelling for GFP, GluA2 and NeuN (Neuronal nuclei). Plots represent quantification analysis of GluA2 signal intensity in HB9:GFP motor neurons at ( U ) E12.5 and ( V ) E17.5. Data represent mean ± SEM, unpaired student t -test performed on n = 4 biological replicates, ∼50 neurons analysed per biological replicate, * P < 0.05. Scale bars 50 μm.
    Figure Legend Snippet: Expression of GluA2 in spinal cords of embryonic SOD1 G93A mice. Cross-sections of lumbar spinal cord from WT (HB9:GFP; WT) and SOD1 G93A (SOD1 G93A ; HB9:GFP) mice at (A–J) E12.5 and ( K–T ) E17.5. Double-immunolabelling for GFP, GluA2 and NeuN (Neuronal nuclei). Plots represent quantification analysis of GluA2 signal intensity in HB9:GFP motor neurons at ( U ) E12.5 and ( V ) E17.5. Data represent mean ± SEM, unpaired student t -test performed on n = 4 biological replicates, ∼50 neurons analysed per biological replicate, * P < 0.05. Scale bars 50 μm.

    Techniques Used: Expressing

    Expression of GRIA2 and ADAR2 in iPSC motor neurons derived from ALS patients with SOD1 mutations and healthy control lines. Representative images of iPSC mature motor neurons derived from ( A–E ) healthy control line and ( F–J ) SOD1 I114T line, immunolabelled with ChAT, GluA2 and TUJ1, counterstained with Hoechst. ( K ) Plot represents quantification analysis of GluA2 signal intensity in iPSC motor neurons. Data represent mean ± SEM, unpaired student t -test performed on n = 3 biological replicates, 50 neurons analysed per biological replicate. (L) Fold change expression of GRIA2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. ( M ) Fold change expression of ADAR2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. Data represent mean ± SEM, n = 3 biological replicates, one-way ANOVA with Dunnett's multiple comparison test, * P < 0.01, ** P < 0.005. Scale bars 50 μm.
    Figure Legend Snippet: Expression of GRIA2 and ADAR2 in iPSC motor neurons derived from ALS patients with SOD1 mutations and healthy control lines. Representative images of iPSC mature motor neurons derived from ( A–E ) healthy control line and ( F–J ) SOD1 I114T line, immunolabelled with ChAT, GluA2 and TUJ1, counterstained with Hoechst. ( K ) Plot represents quantification analysis of GluA2 signal intensity in iPSC motor neurons. Data represent mean ± SEM, unpaired student t -test performed on n = 3 biological replicates, 50 neurons analysed per biological replicate. (L) Fold change expression of GRIA2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. ( M ) Fold change expression of ADAR2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. Data represent mean ± SEM, n = 3 biological replicates, one-way ANOVA with Dunnett's multiple comparison test, * P < 0.01, ** P < 0.005. Scale bars 50 μm.

    Techniques Used: Expressing, Derivative Assay, Quantitative RT-PCR

    agc 005 rrid ab 2039881  (Alomone Labs)


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    Alomone Labs agc 005 rrid ab 2039881
    Agc 005 Rrid Ab 2039881, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    glur2  (Alomone Labs)


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    Alomone Labs glur2
    Astaxanthin inhibits a [Ca 2+ ]i increase in cortical neurons upon ionotropic glutamate receptor activation. ( A – C ) The average [Ca 2+ ]i response in control (black) and AST (red) preincubated neurons stimulated with 50 μM each of NMDA (+ 5 μM glycine), AMPA and KA for 15 min (NMDA: Con n = 23, AST n = 42; AMPA: con n = 27, AST n = 23; KA con n = 30, AST n = 40). ( D ) Dot plot representing the total calcium (area under the curve) after 15 min of NMDA, AMPA and KA stimulation. Arrow heads indicate point of glutamate receptor agonist applications. ( E ) Representative protein expression levels of NMDA (GluN1), AMPA <t>(GluA2)</t> and KA (GluK123) detected by the Western blot analysis with β-actin as the internal reference (individual Western blots figure are provided in ). ( F ) Dot plot indicate the average normalized protein expression for GluN1, GluA2 and GluK123. Data are represented as mean ± SEM from 3–4 different experiments, * p < 0.05. n.s: non-significant.
    Glur2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Astaxanthin Protection against Neuronal Excitotoxicity via Glutamate Receptor Inhibition and Improvement of Mitochondrial Function"

    Article Title: Astaxanthin Protection against Neuronal Excitotoxicity via Glutamate Receptor Inhibition and Improvement of Mitochondrial Function

    Journal: Marine Drugs

    doi: 10.3390/md20100645

    Astaxanthin inhibits a [Ca 2+ ]i increase in cortical neurons upon ionotropic glutamate receptor activation. ( A – C ) The average [Ca 2+ ]i response in control (black) and AST (red) preincubated neurons stimulated with 50 μM each of NMDA (+ 5 μM glycine), AMPA and KA for 15 min (NMDA: Con n = 23, AST n = 42; AMPA: con n = 27, AST n = 23; KA con n = 30, AST n = 40). ( D ) Dot plot representing the total calcium (area under the curve) after 15 min of NMDA, AMPA and KA stimulation. Arrow heads indicate point of glutamate receptor agonist applications. ( E ) Representative protein expression levels of NMDA (GluN1), AMPA (GluA2) and KA (GluK123) detected by the Western blot analysis with β-actin as the internal reference (individual Western blots figure are provided in ). ( F ) Dot plot indicate the average normalized protein expression for GluN1, GluA2 and GluK123. Data are represented as mean ± SEM from 3–4 different experiments, * p < 0.05. n.s: non-significant.
    Figure Legend Snippet: Astaxanthin inhibits a [Ca 2+ ]i increase in cortical neurons upon ionotropic glutamate receptor activation. ( A – C ) The average [Ca 2+ ]i response in control (black) and AST (red) preincubated neurons stimulated with 50 μM each of NMDA (+ 5 μM glycine), AMPA and KA for 15 min (NMDA: Con n = 23, AST n = 42; AMPA: con n = 27, AST n = 23; KA con n = 30, AST n = 40). ( D ) Dot plot representing the total calcium (area under the curve) after 15 min of NMDA, AMPA and KA stimulation. Arrow heads indicate point of glutamate receptor agonist applications. ( E ) Representative protein expression levels of NMDA (GluN1), AMPA (GluA2) and KA (GluK123) detected by the Western blot analysis with β-actin as the internal reference (individual Western blots figure are provided in ). ( F ) Dot plot indicate the average normalized protein expression for GluN1, GluA2 and GluK123. Data are represented as mean ± SEM from 3–4 different experiments, * p < 0.05. n.s: non-significant.

    Techniques Used: Activation Assay, Expressing, Western Blot

    glur2  (Alomone Labs)


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    Alomone Labs glur2
    a-c , Synaptic vesicles were labeled live using an antibody against a luminal epitope of synaptotagmin 1 (Syt1, magenta). The vesicular glutamate transporter (vGluT1, blue) and PSD95 (gray) were immunostained using an antibody and a nanobody, respectively. a , Recently endocytosed vesicle exhibiting circular morphology. b , Readily retrievable pool molecules form patches containing Syt1/vGluT1 (top), which are dispersed by cholesterol extraction using MβCD (bottom). c , MβCD causes molecules to spread across larger areas (left: N = 22-19, 2 independent experiments, p < 0.0044, Mann-Whitney test; right: N = 22-22, 2 independent experiments, p = 0.8937), although the signal per vesicle (the Syt1 copy number) remains unchanged. d , A visualization of PSDs (top and side views), after immunostaining PSD95 with the same nanobody used in a-c, and Shank2 and Homer1 with specific antibodies. The graph indicates the axial positioning, which agrees well with the literature . N = 11 measurements for each protein, 2 independent experiments; symbols show the medians, SEM and SD. e , Side view of a postsynapse displaying PSD95, MAP2 and two glutamate receptors <t>(GluR2,</t> AMPA type, and GluN2b, NMDA type). f , ONE images of PSD95 (top views), before or after the addition of 10% 1,6-hexanediol (Hex). g , Line scans through the PSD95 stainings shown in panel f. h , An analysis of PSD95 spot profiles; N = 10-7 synapses, Friedman test followed by Dunn-Sidak testing, p = 0.0027; the error bars show the SEM. For details on the analysis, see .
    Glur2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    1) Product Images from "Expansion microscopy at one nanometer resolution"

    Article Title: Expansion microscopy at one nanometer resolution

    Journal: bioRxiv

    doi: 10.1101/2022.08.03.502284

    a-c , Synaptic vesicles were labeled live using an antibody against a luminal epitope of synaptotagmin 1 (Syt1, magenta). The vesicular glutamate transporter (vGluT1, blue) and PSD95 (gray) were immunostained using an antibody and a nanobody, respectively. a , Recently endocytosed vesicle exhibiting circular morphology. b , Readily retrievable pool molecules form patches containing Syt1/vGluT1 (top), which are dispersed by cholesterol extraction using MβCD (bottom). c , MβCD causes molecules to spread across larger areas (left: N = 22-19, 2 independent experiments, p < 0.0044, Mann-Whitney test; right: N = 22-22, 2 independent experiments, p = 0.8937), although the signal per vesicle (the Syt1 copy number) remains unchanged. d , A visualization of PSDs (top and side views), after immunostaining PSD95 with the same nanobody used in a-c, and Shank2 and Homer1 with specific antibodies. The graph indicates the axial positioning, which agrees well with the literature . N = 11 measurements for each protein, 2 independent experiments; symbols show the medians, SEM and SD. e , Side view of a postsynapse displaying PSD95, MAP2 and two glutamate receptors (GluR2, AMPA type, and GluN2b, NMDA type). f , ONE images of PSD95 (top views), before or after the addition of 10% 1,6-hexanediol (Hex). g , Line scans through the PSD95 stainings shown in panel f. h , An analysis of PSD95 spot profiles; N = 10-7 synapses, Friedman test followed by Dunn-Sidak testing, p = 0.0027; the error bars show the SEM. For details on the analysis, see .
    Figure Legend Snippet: a-c , Synaptic vesicles were labeled live using an antibody against a luminal epitope of synaptotagmin 1 (Syt1, magenta). The vesicular glutamate transporter (vGluT1, blue) and PSD95 (gray) were immunostained using an antibody and a nanobody, respectively. a , Recently endocytosed vesicle exhibiting circular morphology. b , Readily retrievable pool molecules form patches containing Syt1/vGluT1 (top), which are dispersed by cholesterol extraction using MβCD (bottom). c , MβCD causes molecules to spread across larger areas (left: N = 22-19, 2 independent experiments, p < 0.0044, Mann-Whitney test; right: N = 22-22, 2 independent experiments, p = 0.8937), although the signal per vesicle (the Syt1 copy number) remains unchanged. d , A visualization of PSDs (top and side views), after immunostaining PSD95 with the same nanobody used in a-c, and Shank2 and Homer1 with specific antibodies. The graph indicates the axial positioning, which agrees well with the literature . N = 11 measurements for each protein, 2 independent experiments; symbols show the medians, SEM and SD. e , Side view of a postsynapse displaying PSD95, MAP2 and two glutamate receptors (GluR2, AMPA type, and GluN2b, NMDA type). f , ONE images of PSD95 (top views), before or after the addition of 10% 1,6-hexanediol (Hex). g , Line scans through the PSD95 stainings shown in panel f. h , An analysis of PSD95 spot profiles; N = 10-7 synapses, Friedman test followed by Dunn-Sidak testing, p = 0.0027; the error bars show the SEM. For details on the analysis, see .

    Techniques Used: Labeling, MANN-WHITNEY, Immunostaining

    polyclonal rabbit glua2 antibody  (Alomone Labs)


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    Alomone Labs polyclonal rabbit glua2 antibody
    Polyclonal Rabbit Glua2 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    glur2  (Alomone Labs)


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    Alomone Labs glur2
    MT treatment reduced IHC ribbon synapse loss and ANF degeneration induced by acoustic trauma. (A) Representative images of the IHC ribbon synapse at approximately 16–20 kHz region of the cochlea 14d after the noise exposure. Cochlear surface preparations were immunolabelled with CtBP2 (red), <t>GluA2</t> (green) and Myosin VIIa (blue). (B) Quantification analysis of paired synapse per IHC, indicated by the juxtaposition of CtBP2 and GluA2. (C) Representative images of ANF and HCs in middle turn area of the midmodiolar sections 14d after the noise exposure. Cochlear sections were immunolabelled with Tuj1 (red) and Myosin VIIa (green). (D) Quantification analysis of ANF density 14d after the noise exposure. Data are presented as means ± SD, n = 4 for each group, *** p < 0.001 vs. control group, # p < 0.05 vs. vehicle-treated group, ### p < 0.001 vs. vehicle-treated group. IHC, inner hair cell; ANF, auditory nerve fiber.
    Glur2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glur2/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
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    Images

    1) Product Images from "mito-TEMPO Attenuates Oxidative Stress and Mitochondrial Dysfunction in Noise-Induced Hearing Loss via Maintaining TFAM-mtDNA Interaction and Mitochondrial Biogenesis"

    Article Title: mito-TEMPO Attenuates Oxidative Stress and Mitochondrial Dysfunction in Noise-Induced Hearing Loss via Maintaining TFAM-mtDNA Interaction and Mitochondrial Biogenesis

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2022.803718

    MT treatment reduced IHC ribbon synapse loss and ANF degeneration induced by acoustic trauma. (A) Representative images of the IHC ribbon synapse at approximately 16–20 kHz region of the cochlea 14d after the noise exposure. Cochlear surface preparations were immunolabelled with CtBP2 (red), GluA2 (green) and Myosin VIIa (blue). (B) Quantification analysis of paired synapse per IHC, indicated by the juxtaposition of CtBP2 and GluA2. (C) Representative images of ANF and HCs in middle turn area of the midmodiolar sections 14d after the noise exposure. Cochlear sections were immunolabelled with Tuj1 (red) and Myosin VIIa (green). (D) Quantification analysis of ANF density 14d after the noise exposure. Data are presented as means ± SD, n = 4 for each group, *** p < 0.001 vs. control group, # p < 0.05 vs. vehicle-treated group, ### p < 0.001 vs. vehicle-treated group. IHC, inner hair cell; ANF, auditory nerve fiber.
    Figure Legend Snippet: MT treatment reduced IHC ribbon synapse loss and ANF degeneration induced by acoustic trauma. (A) Representative images of the IHC ribbon synapse at approximately 16–20 kHz region of the cochlea 14d after the noise exposure. Cochlear surface preparations were immunolabelled with CtBP2 (red), GluA2 (green) and Myosin VIIa (blue). (B) Quantification analysis of paired synapse per IHC, indicated by the juxtaposition of CtBP2 and GluA2. (C) Representative images of ANF and HCs in middle turn area of the midmodiolar sections 14d after the noise exposure. Cochlear sections were immunolabelled with Tuj1 (red) and Myosin VIIa (green). (D) Quantification analysis of ANF density 14d after the noise exposure. Data are presented as means ± SD, n = 4 for each group, *** p < 0.001 vs. control group, # p < 0.05 vs. vehicle-treated group, ### p < 0.001 vs. vehicle-treated group. IHC, inner hair cell; ANF, auditory nerve fiber.

    Techniques Used:

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    a Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled for surface <t>GluA2</t> (under non-permeabilizing conditions). Graph showing the number of surface GluA2 labeling, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA, F (2, 15) = 38.28) (Tukey’s test P control-NMDAR < 0.0001, P control-mGluR < 0.0001, P NMDAR-mGluR = 0.8438). b Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled with an antibody against endogenous LC3 (autophagic structures) and MAP2 (dendrites). Graph showing the number of dendritic LC3-positive puncta in secondary dendrites, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F2,24) = 15.11, P < 0.0001) (Tukey’s test Pcontrol-NMDA = 0.0005, P control-mGluR = 0.0001). c Same as in b , but neurons were pretreated for 1 h before, during and after the pulse with wortmannin (500 nM) or SBI-0206965 (500 nM). Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions (U: untreated, W: wortmannin, S: SBI-0206965). Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(8,45) = 33.83, P < 0.0001) (Tukey’s test P control/S-NMDA/S = 0.3677, P control/W-NMDA/W = 0.9986, P NMDA/U-NMDA/W < 0.0001, P NMDA/U-NMDA/S < 0.0001, P control/S-DHPG/S = 0.9674, P control/W-DHPG/W = 0.9989, P DHPG/U-DHPG/W < 0.0001, P DHPG/U-DHPG/S < 0.0001). d Same as in b with neurons that were infected with AAV plasmids carrying 4 shRNA sequences against atg5 ( sh-atg5 ) or scrambled control ( sh-scramble ), under the CamK2a promoter. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(5,30) = 16.94, P < 0.0001) (Tukey’s test P control/scr-control/atg5 = 0.9999, P NMDA/scr-NMDA/atg5 = 0.0025, P DHPG/scr-DHPG/atg5 < 0.0001, P control/scr-NMDA/scr < 0.0001, P control/scr-DGPG/scr < 0.0001, P control/atg5-NMDA/atg5 = 0.8959, P control/atg5-DHPG/atg5 = 0.9637). e Same as in b , but neurons were immunolabeled 15 min after NMDAR- and mGluR-LTD and treated for 1 h before, during and after the pulse with Ifenprodil (10 μM) or MTEP (10 μM) and JNJ16259685 (10 μM) to pharmacologically inhibit NR2B and mGluR1/5 receptors, respectively. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F (3,32) = 74.46, P < 0.0001) (Tukey’s test, P NMDA-NMDA+IFE < 0.0001, P DHPG-DHPG+MTEP/JNJ < 0.0001). Scale bars: 10 μm for all panels.
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    Images

    1) Product Images from "Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice"

    Article Title: Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice

    Journal: Nature Communications

    doi: 10.1038/s41467-022-28301-z

    a Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled for surface GluA2 (under non-permeabilizing conditions). Graph showing the number of surface GluA2 labeling, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA, F (2, 15) = 38.28) (Tukey’s test P control-NMDAR < 0.0001, P control-mGluR < 0.0001, P NMDAR-mGluR = 0.8438). b Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled with an antibody against endogenous LC3 (autophagic structures) and MAP2 (dendrites). Graph showing the number of dendritic LC3-positive puncta in secondary dendrites, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F2,24) = 15.11, P < 0.0001) (Tukey’s test Pcontrol-NMDA = 0.0005, P control-mGluR = 0.0001). c Same as in b , but neurons were pretreated for 1 h before, during and after the pulse with wortmannin (500 nM) or SBI-0206965 (500 nM). Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions (U: untreated, W: wortmannin, S: SBI-0206965). Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(8,45) = 33.83, P < 0.0001) (Tukey’s test P control/S-NMDA/S = 0.3677, P control/W-NMDA/W = 0.9986, P NMDA/U-NMDA/W < 0.0001, P NMDA/U-NMDA/S < 0.0001, P control/S-DHPG/S = 0.9674, P control/W-DHPG/W = 0.9989, P DHPG/U-DHPG/W < 0.0001, P DHPG/U-DHPG/S < 0.0001). d Same as in b with neurons that were infected with AAV plasmids carrying 4 shRNA sequences against atg5 ( sh-atg5 ) or scrambled control ( sh-scramble ), under the CamK2a promoter. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(5,30) = 16.94, P < 0.0001) (Tukey’s test P control/scr-control/atg5 = 0.9999, P NMDA/scr-NMDA/atg5 = 0.0025, P DHPG/scr-DHPG/atg5 < 0.0001, P control/scr-NMDA/scr < 0.0001, P control/scr-DGPG/scr < 0.0001, P control/atg5-NMDA/atg5 = 0.8959, P control/atg5-DHPG/atg5 = 0.9637). e Same as in b , but neurons were immunolabeled 15 min after NMDAR- and mGluR-LTD and treated for 1 h before, during and after the pulse with Ifenprodil (10 μM) or MTEP (10 μM) and JNJ16259685 (10 μM) to pharmacologically inhibit NR2B and mGluR1/5 receptors, respectively. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F (3,32) = 74.46, P < 0.0001) (Tukey’s test, P NMDA-NMDA+IFE < 0.0001, P DHPG-DHPG+MTEP/JNJ < 0.0001). Scale bars: 10 μm for all panels.
    Figure Legend Snippet: a Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled for surface GluA2 (under non-permeabilizing conditions). Graph showing the number of surface GluA2 labeling, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA, F (2, 15) = 38.28) (Tukey’s test P control-NMDAR < 0.0001, P control-mGluR < 0.0001, P NMDAR-mGluR = 0.8438). b Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled with an antibody against endogenous LC3 (autophagic structures) and MAP2 (dendrites). Graph showing the number of dendritic LC3-positive puncta in secondary dendrites, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F2,24) = 15.11, P < 0.0001) (Tukey’s test Pcontrol-NMDA = 0.0005, P control-mGluR = 0.0001). c Same as in b , but neurons were pretreated for 1 h before, during and after the pulse with wortmannin (500 nM) or SBI-0206965 (500 nM). Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions (U: untreated, W: wortmannin, S: SBI-0206965). Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(8,45) = 33.83, P < 0.0001) (Tukey’s test P control/S-NMDA/S = 0.3677, P control/W-NMDA/W = 0.9986, P NMDA/U-NMDA/W < 0.0001, P NMDA/U-NMDA/S < 0.0001, P control/S-DHPG/S = 0.9674, P control/W-DHPG/W = 0.9989, P DHPG/U-DHPG/W < 0.0001, P DHPG/U-DHPG/S < 0.0001). d Same as in b with neurons that were infected with AAV plasmids carrying 4 shRNA sequences against atg5 ( sh-atg5 ) or scrambled control ( sh-scramble ), under the CamK2a promoter. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(5,30) = 16.94, P < 0.0001) (Tukey’s test P control/scr-control/atg5 = 0.9999, P NMDA/scr-NMDA/atg5 = 0.0025, P DHPG/scr-DHPG/atg5 < 0.0001, P control/scr-NMDA/scr < 0.0001, P control/scr-DGPG/scr < 0.0001, P control/atg5-NMDA/atg5 = 0.8959, P control/atg5-DHPG/atg5 = 0.9637). e Same as in b , but neurons were immunolabeled 15 min after NMDAR- and mGluR-LTD and treated for 1 h before, during and after the pulse with Ifenprodil (10 μM) or MTEP (10 μM) and JNJ16259685 (10 μM) to pharmacologically inhibit NR2B and mGluR1/5 receptors, respectively. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F (3,32) = 74.46, P < 0.0001) (Tukey’s test, P NMDA-NMDA+IFE < 0.0001, P DHPG-DHPG+MTEP/JNJ < 0.0001). Scale bars: 10 μm for all panels.

    Techniques Used: Cell Culture, Immunolabeling, Labeling, Infection, shRNA

    a Confocal images of dendrites immunolabeled with an antibody against the extracellular region of GluA2 under control conditions or 15 min after LTD induction and in the absence or presence of Dynamin-1 inhibitory peptide (50 µM) or SBI-0206965 (500 nM), a selective inhibitor of the ULK1 kinase activity. Inhibitors were applied 25 min before, during and 15 min after the pulses. Scale bar: 10 µm. Graph showing the surface labeling of GluA2, normalized to dendritic length under the aforementioned conditions. Bars represent mean values ± SEM. N = 9 independent experiments. Statistical analysis was performed using one-way ANOVA (F (8, 72) = 7.411, P < 0.0001) (Tukey’s test P control-control/D > 0.99, P control-control/S = 0.9971, P NMDA-NMDA/D = 0.0451, P NMDA-NMDA/S = 0.0008, P DHPG-DHPG/D = 0.0017, P DHPG-DHPG/S = 0.0002). b Confocal images of dendrites of neurons expressing 4 scrambled sequences ( sh-scramble ), or 4 sh-RNAs against atg5 ( sh-atg5 ), immunolabeled with an antibody against the extracellular region of GluA2 under control conditions or 15 min after LTD induction. Graph showing the surface labeling of GluA2, normalized to dendritic length under the aforementioned conditions. Bars represent mean values ± SEM. N = 10 independent experiments. Statistical analysis was performed using one-way ANOVA (F (5, 54) = 30.02, P < 0.0001) (Tukey’s test, P control/scr-control/atg5 = 0.0626, P NMDA/scr-NMDA/atg5 < 0.0001, P DHPG/scr-DHPG/atg5 < 0.0001, P control/atg5-NMDA/atg5 > 0.99, P control/atg5-DHPG/atg5 = 0.8602, P control/scr-NMDA/scr = 0.0008, P control/scr-DHPG/scr < 0.0001). c Representative images of consecutive confocal z-planes of cultured neurons immunostained with antibodies against PSD95, LC3, and MAP2 to label the dendrites, 15 min after cLTD. Note the colocalization of PSD95 and LC3 in dendritic spines (yellow arrows) and in the dendritic shaft (white arrows), in consecutive z-planes. Scale bar: 10 µm. Graph showing the percentage of PSD95 puncta co-localizing with LC3 in consecutive confocal z-planes in dendritic spines and shafts in control neurons or 15 min after chemically induced NMDAR- or mGluR-LTD. Bars represent mean values ± SEM. N = 8 independent experiments. Statistical analysis was performed by one-way ANOVA (F(5,42) = 48.43, P < 0.0001) (Tukey’s test for dendritic shaft, P control-NMDA = 0.0569, P control-DHPG = 0.1948, for dendritic spines, P control-NMDA < 0.0001, P control-DHPG < 0.0001). d Western blot analysis for GluA2 and PSD95 in lysates of cultured neurons in control conditions or 15 min after NMDAR- and mGluR-LTD and in the presence or absence of Bafilomycin A1 (50 µM) for 15 min before, during, and 15 min after the NMDA and DHPG pulses. e Western blot analysis for GluA2 and PSD95 in lysates of cultured neurons in control conditions or 15 min after NMDAR- and mGluR-LTD and in the presence or absence of SBI-0206965 (500 nM) for 30 min before, during, and 15 min after the NMDA and DHPG pulses. f Western blot analysis for GluA2 and PSD95 in lysates of cultured shscrambled or sh-atg5 expressing neurons in control conditions or 15 min after NMDAR- and mGluR-LTD. d – f Graphs showing the levels of PSD95 and GluA2 levels in the indicated conditions, normalized to total protein levels. Bars represent mean values ± SEM. Statistical analysis was performed by one-way ANOVA. d (N = 9 independent experiments) PSD95: F(5,48) = 15.08, P < 0.0001 (Tukey’s test P control-control/Baf = 0.7566, P control-NMDA = 0.0016, P control-DHPG = 0.0081, P NMDA-NMDA/Baf < 0.0001, P DHPG-DHPG/Baf = 0.0013. GluA2: F(5,48)=6.627, P < 0.0001 (Tukey’s test P control-control/Baf = 0.9692, P control-NMDA = 0.0014, P control-DHPG = 0.0067, P NMDA-NMDA/Baf = 0.0421, P DHPG-DHPG/Baf = 0.0127. e ( N = 7 independent experiments) PSD95: F(5,36) = 23.80, P < 0.0001. (Tukey’s test P control-control/SBI > 0.99, P NMDA-NMDA/SBI < 0.0001, P DHPG-DHPG/SBI < 0.0001, P control-NMDA < 0.0001, P control-DHPG < 0.0001, P control/SBI-NMDA/SBI = 0.9764, P control/SBI-DHPG/SBI = 0.6286). Panel e, GluA2: F(5,36)=11.73, P < 0.0001. (Tukey’s test P control-control/SBI = 0.9179, P NMDA-NMDA/SBI = 0.0001, P DHPG-DHPG/SBI = 0.0002, P control-NMDA = 0.0099, P control-DHPG = 0.0323, P control/SBI-NMDA/SBI = 0.9959, P control/SBI-DHPG/SBI = 0.9407). f ( N = 7 independent experiments) PSD95: F(5,36) = 10.93, P < 0.0001. (Tukey’s test P control/scr-control/atg5 = 0.7927, P NMDA/scr-NMDA/atg5 = 0.0045, P DHPG/scr-DHPG/atg5 = 0.0003, P control/scr-NMDA/scr = 0.0134, P control/scr-DHPG/scr = 0.0030, P control/atg5-NMDA/atg5 = 0.9488, P control/atg5-DHPG/atg5 = 0.9976). GluA2: F(5,36) = 10.79, P < 0.0001. (Tukey’s test P control/scr-control/atg5 > 0.99, P NMDA/scr-NMDA/atg5 = 0,0001, P DHPG/scr-DHPG/atg5 = 0.0019, P control/scr-NMDA/scr = 0.0134, P control/scr-DHPG/scr = 0.0021, P control/atg5-NMDA/atg5 = 0.5844, P control/atg5-DHPG/atg5 > 0.99).
    Figure Legend Snippet: a Confocal images of dendrites immunolabeled with an antibody against the extracellular region of GluA2 under control conditions or 15 min after LTD induction and in the absence or presence of Dynamin-1 inhibitory peptide (50 µM) or SBI-0206965 (500 nM), a selective inhibitor of the ULK1 kinase activity. Inhibitors were applied 25 min before, during and 15 min after the pulses. Scale bar: 10 µm. Graph showing the surface labeling of GluA2, normalized to dendritic length under the aforementioned conditions. Bars represent mean values ± SEM. N = 9 independent experiments. Statistical analysis was performed using one-way ANOVA (F (8, 72) = 7.411, P < 0.0001) (Tukey’s test P control-control/D > 0.99, P control-control/S = 0.9971, P NMDA-NMDA/D = 0.0451, P NMDA-NMDA/S = 0.0008, P DHPG-DHPG/D = 0.0017, P DHPG-DHPG/S = 0.0002). b Confocal images of dendrites of neurons expressing 4 scrambled sequences ( sh-scramble ), or 4 sh-RNAs against atg5 ( sh-atg5 ), immunolabeled with an antibody against the extracellular region of GluA2 under control conditions or 15 min after LTD induction. Graph showing the surface labeling of GluA2, normalized to dendritic length under the aforementioned conditions. Bars represent mean values ± SEM. N = 10 independent experiments. Statistical analysis was performed using one-way ANOVA (F (5, 54) = 30.02, P < 0.0001) (Tukey’s test, P control/scr-control/atg5 = 0.0626, P NMDA/scr-NMDA/atg5 < 0.0001, P DHPG/scr-DHPG/atg5 < 0.0001, P control/atg5-NMDA/atg5 > 0.99, P control/atg5-DHPG/atg5 = 0.8602, P control/scr-NMDA/scr = 0.0008, P control/scr-DHPG/scr < 0.0001). c Representative images of consecutive confocal z-planes of cultured neurons immunostained with antibodies against PSD95, LC3, and MAP2 to label the dendrites, 15 min after cLTD. Note the colocalization of PSD95 and LC3 in dendritic spines (yellow arrows) and in the dendritic shaft (white arrows), in consecutive z-planes. Scale bar: 10 µm. Graph showing the percentage of PSD95 puncta co-localizing with LC3 in consecutive confocal z-planes in dendritic spines and shafts in control neurons or 15 min after chemically induced NMDAR- or mGluR-LTD. Bars represent mean values ± SEM. N = 8 independent experiments. Statistical analysis was performed by one-way ANOVA (F(5,42) = 48.43, P < 0.0001) (Tukey’s test for dendritic shaft, P control-NMDA = 0.0569, P control-DHPG = 0.1948, for dendritic spines, P control-NMDA < 0.0001, P control-DHPG < 0.0001). d Western blot analysis for GluA2 and PSD95 in lysates of cultured neurons in control conditions or 15 min after NMDAR- and mGluR-LTD and in the presence or absence of Bafilomycin A1 (50 µM) for 15 min before, during, and 15 min after the NMDA and DHPG pulses. e Western blot analysis for GluA2 and PSD95 in lysates of cultured neurons in control conditions or 15 min after NMDAR- and mGluR-LTD and in the presence or absence of SBI-0206965 (500 nM) for 30 min before, during, and 15 min after the NMDA and DHPG pulses. f Western blot analysis for GluA2 and PSD95 in lysates of cultured shscrambled or sh-atg5 expressing neurons in control conditions or 15 min after NMDAR- and mGluR-LTD. d – f Graphs showing the levels of PSD95 and GluA2 levels in the indicated conditions, normalized to total protein levels. Bars represent mean values ± SEM. Statistical analysis was performed by one-way ANOVA. d (N = 9 independent experiments) PSD95: F(5,48) = 15.08, P < 0.0001 (Tukey’s test P control-control/Baf = 0.7566, P control-NMDA = 0.0016, P control-DHPG = 0.0081, P NMDA-NMDA/Baf < 0.0001, P DHPG-DHPG/Baf = 0.0013. GluA2: F(5,48)=6.627, P < 0.0001 (Tukey’s test P control-control/Baf = 0.9692, P control-NMDA = 0.0014, P control-DHPG = 0.0067, P NMDA-NMDA/Baf = 0.0421, P DHPG-DHPG/Baf = 0.0127. e ( N = 7 independent experiments) PSD95: F(5,36) = 23.80, P < 0.0001. (Tukey’s test P control-control/SBI > 0.99, P NMDA-NMDA/SBI < 0.0001, P DHPG-DHPG/SBI < 0.0001, P control-NMDA < 0.0001, P control-DHPG < 0.0001, P control/SBI-NMDA/SBI = 0.9764, P control/SBI-DHPG/SBI = 0.6286). Panel e, GluA2: F(5,36)=11.73, P < 0.0001. (Tukey’s test P control-control/SBI = 0.9179, P NMDA-NMDA/SBI = 0.0001, P DHPG-DHPG/SBI = 0.0002, P control-NMDA = 0.0099, P control-DHPG = 0.0323, P control/SBI-NMDA/SBI = 0.9959, P control/SBI-DHPG/SBI = 0.9407). f ( N = 7 independent experiments) PSD95: F(5,36) = 10.93, P < 0.0001. (Tukey’s test P control/scr-control/atg5 = 0.7927, P NMDA/scr-NMDA/atg5 = 0.0045, P DHPG/scr-DHPG/atg5 = 0.0003, P control/scr-NMDA/scr = 0.0134, P control/scr-DHPG/scr = 0.0030, P control/atg5-NMDA/atg5 = 0.9488, P control/atg5-DHPG/atg5 = 0.9976). GluA2: F(5,36) = 10.79, P < 0.0001. (Tukey’s test P control/scr-control/atg5 > 0.99, P NMDA/scr-NMDA/atg5 = 0,0001, P DHPG/scr-DHPG/atg5 = 0.0019, P control/scr-NMDA/scr = 0.0134, P control/scr-DHPG/scr = 0.0021, P control/atg5-NMDA/atg5 = 0.5844, P control/atg5-DHPG/atg5 > 0.99).

    Techniques Used: Immunolabeling, Activity Assay, Labeling, Expressing, Cell Culture, Western Blot

    a – d Western blot analyses of different fractions along the autophagic vesicle purification procedure, using antibodies against a autophagosomal markers (LC3B, p62, Atg16L1, and Atg9A), b ER-Golgi markers (TGN, LMAN1, SAR1a), c endosomal markers (Rab11b, EEA1), and d markers of the plasma-membrane (Stx4), extracellular vesicles (Alix) and nuclear extracts (TBP). N = 3 independent experiments. e Graph showing the cell component analysis, as false discovery rate (FDR)-corrected p -values, of the dynamic cargo (total of 393 proteins) that is enriched (up) or less abundant (down) in AVs after LTD, compared to control. f Graphical representation of proteins enriched in AVs upon LTD, with relation to the synapse. g Western blot analysis of PK-treated control and LTD-AVs, validating the presence of the proteins identified by the proteomic analyses in the autophagic vesicles. Postsynaptic density (PSD) fraction was used as reference. Graph showing the fold change of the normalized levels of the proteins validated by western blot, as a ratio of LTD to control. Cargo proteins were normalized to the levels of p62, which remains unaffected at the early phase of LTD. N = 3 independent AV preparations. Bars represent mean values ± SEM. Statistical analysis was performed using paired, two-tailed Student’s t -test (GluA1, N = 6, P = 0.0002; GluA2, N = 6, P = 0.0039; Pick1, N = 5, P = 0.011; SAP97, N = 5, P = 0.0179; FYN, N = 8, P < 0.0001; CamKIIa, N = 8, P < 0.0001; IL1RAPL1, N = 8, P = 0.0004; Adam22, N = 4, P = 0.0018; INA, N = 3, P = 0.0287; MYH10, N = 8, P < 0.0001; ITPKA, N = 6, P = 0.0006; KCC2, N = 4, P = 0. 0352; cofilin-1, N = 6, P = 0.005; dynamin, N = 6, P = 0.0005; p62, N = 6, P = 0.9809). All indicated molecular weights in a – d and g are in kDaltons (kD).
    Figure Legend Snippet: a – d Western blot analyses of different fractions along the autophagic vesicle purification procedure, using antibodies against a autophagosomal markers (LC3B, p62, Atg16L1, and Atg9A), b ER-Golgi markers (TGN, LMAN1, SAR1a), c endosomal markers (Rab11b, EEA1), and d markers of the plasma-membrane (Stx4), extracellular vesicles (Alix) and nuclear extracts (TBP). N = 3 independent experiments. e Graph showing the cell component analysis, as false discovery rate (FDR)-corrected p -values, of the dynamic cargo (total of 393 proteins) that is enriched (up) or less abundant (down) in AVs after LTD, compared to control. f Graphical representation of proteins enriched in AVs upon LTD, with relation to the synapse. g Western blot analysis of PK-treated control and LTD-AVs, validating the presence of the proteins identified by the proteomic analyses in the autophagic vesicles. Postsynaptic density (PSD) fraction was used as reference. Graph showing the fold change of the normalized levels of the proteins validated by western blot, as a ratio of LTD to control. Cargo proteins were normalized to the levels of p62, which remains unaffected at the early phase of LTD. N = 3 independent AV preparations. Bars represent mean values ± SEM. Statistical analysis was performed using paired, two-tailed Student’s t -test (GluA1, N = 6, P = 0.0002; GluA2, N = 6, P = 0.0039; Pick1, N = 5, P = 0.011; SAP97, N = 5, P = 0.0179; FYN, N = 8, P < 0.0001; CamKIIa, N = 8, P < 0.0001; IL1RAPL1, N = 8, P = 0.0004; Adam22, N = 4, P = 0.0018; INA, N = 3, P = 0.0287; MYH10, N = 8, P < 0.0001; ITPKA, N = 6, P = 0.0006; KCC2, N = 4, P = 0. 0352; cofilin-1, N = 6, P = 0.005; dynamin, N = 6, P = 0.0005; p62, N = 6, P = 0.9809). All indicated molecular weights in a – d and g are in kDaltons (kD).

    Techniques Used: Western Blot, Purification, Two Tailed Test


    Figure Legend Snippet:

    Techniques Used: Concentration Assay, Activity Assay, Plasmid Preparation, Avidin-Biotin Assay, Infection, In Vivo

    glur2 ampar antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs glur2 ampar antibody
    Characterization of differentiated PC12 cells and validation of single-molecule labeling. (A,B) Left, Intensity profiles of a single ATTO 488-labeled <t>GluR2-AMPAR</t> (A) and mGluR1 (B) signal. The arrows indicate single-step photobleaching. Right, Histogram showing the intensity value of every spot found in a recording of ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) , superimposed with a single fitted lognormal curve (blue line). (C) Representative trajectories of AMPAR molecules on somas and neurites. Scale bar = 2 μm. (D) The mean square displacement functions and trajectories represent AMPAR molecules with Brownian motion (red) and confined motion (blue). Scale bar = 0.1 μm. (E,F) The cumulative probability functions of D values of AMPAR (E) and mGluR1 (F) on neurites and somas ( n = 510–676 trajectories). *** p < 0.001.
    Glur2 Ampar Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons"

    Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2021.708715

    Characterization of differentiated PC12 cells and validation of single-molecule labeling. (A,B) Left, Intensity profiles of a single ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) signal. The arrows indicate single-step photobleaching. Right, Histogram showing the intensity value of every spot found in a recording of ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) , superimposed with a single fitted lognormal curve (blue line). (C) Representative trajectories of AMPAR molecules on somas and neurites. Scale bar = 2 μm. (D) The mean square displacement functions and trajectories represent AMPAR molecules with Brownian motion (red) and confined motion (blue). Scale bar = 0.1 μm. (E,F) The cumulative probability functions of D values of AMPAR (E) and mGluR1 (F) on neurites and somas ( n = 510–676 trajectories). *** p < 0.001.
    Figure Legend Snippet: Characterization of differentiated PC12 cells and validation of single-molecule labeling. (A,B) Left, Intensity profiles of a single ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) signal. The arrows indicate single-step photobleaching. Right, Histogram showing the intensity value of every spot found in a recording of ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) , superimposed with a single fitted lognormal curve (blue line). (C) Representative trajectories of AMPAR molecules on somas and neurites. Scale bar = 2 μm. (D) The mean square displacement functions and trajectories represent AMPAR molecules with Brownian motion (red) and confined motion (blue). Scale bar = 0.1 μm. (E,F) The cumulative probability functions of D values of AMPAR (E) and mGluR1 (F) on neurites and somas ( n = 510–676 trajectories). *** p < 0.001.

    Techniques Used: Labeling

    Effect of E2 on the surface movement of GluR2-AMPAR and mGluR1. (A) Effect of different concentrations of E2 on the diffusion coefficient (D, μm 2 /s) of GluR2-AMPAR (A) and mGluR1 (B) (% of vehicle treatment as the mean ± SEM, n = 425–1145 trajectories per group). (C,D) Line graphs depict changes in D of GluR2-AMPAR (C) and mGluR1 (D) molecules at different time points after the administration of the most effective concentration of E2 (% of vehicle treatment as the mean D ± SEM, n = 117–187 trajectories per time point). * p < 0.05; ** p < 0.01; *** p < 0.001.
    Figure Legend Snippet: Effect of E2 on the surface movement of GluR2-AMPAR and mGluR1. (A) Effect of different concentrations of E2 on the diffusion coefficient (D, μm 2 /s) of GluR2-AMPAR (A) and mGluR1 (B) (% of vehicle treatment as the mean ± SEM, n = 425–1145 trajectories per group). (C,D) Line graphs depict changes in D of GluR2-AMPAR (C) and mGluR1 (D) molecules at different time points after the administration of the most effective concentration of E2 (% of vehicle treatment as the mean D ± SEM, n = 117–187 trajectories per time point). * p < 0.05; ** p < 0.01; *** p < 0.001.

    Techniques Used: Diffusion-based Assay, Concentration Assay

    Effect of estrogen receptor modulation on the surface movement of GluR2-AMPAR. (A) Representative PCR gel electrophoresis image depicting the expression of estrogen receptor beta (ERβ) and G protein-coupled estrogen receptor 1 (GPER1) mRNA in dPC12. Estrogen receptor alpha (ERα) mRNA was not detected. (B) Histograms demonstrate the mean D AMPAR as a percentage of vehicle control on somas and neurites in the presence of the estrogen receptor, β (ERβ) agonist diarylpropionitrile (DPN), a GPER1 agonist (G1), G1+DPN together, a GPER1 antagonist (G15) and G15+E2 (with 100 pM of E2 on the somas and 100 nM of E2 on the neurites) (mean ± SEM; n = 215–641 trajectories). *** p < 0.001.
    Figure Legend Snippet: Effect of estrogen receptor modulation on the surface movement of GluR2-AMPAR. (A) Representative PCR gel electrophoresis image depicting the expression of estrogen receptor beta (ERβ) and G protein-coupled estrogen receptor 1 (GPER1) mRNA in dPC12. Estrogen receptor alpha (ERα) mRNA was not detected. (B) Histograms demonstrate the mean D AMPAR as a percentage of vehicle control on somas and neurites in the presence of the estrogen receptor, β (ERβ) agonist diarylpropionitrile (DPN), a GPER1 agonist (G1), G1+DPN together, a GPER1 antagonist (G15) and G15+E2 (with 100 pM of E2 on the somas and 100 nM of E2 on the neurites) (mean ± SEM; n = 215–641 trajectories). *** p < 0.001.

    Techniques Used: Nucleic Acid Electrophoresis, Expressing

    The GluR2-AMPAR/GPER1 ratio and molecular distance between GPER1 and GluR2-AMPAR in the membrane. (A) STORM images depicting immunolabeled AMPAR (magenta) and GPER1 (cyan) molecules on dPC12. Dashed lines delineate the borders of the neurites and somas. Scale bar = 2 μm; inset Scale bar = 0.5 μm. (B) The ratio between the number of GPER1 and AMPAR molecules (GPER1/GluR2-AMPAR) on the neurites and somas ( n = 11 somas or neurites). (C1) Photomicrographs depict GPER1 immunoreactivity (visualized with STED microscopy) in dPC12 after 10 min of vehicle (left) or of 100 nM of E2 treatment (right). Scale bar = 2 μm. (C2) One 2 μm 2 (between parallel white bars) and one 10 μm 2 (to the left) areas were selected within each ROI for the membrane and cytoplasmic regions of each cell, respectively. Integrated density was calculated and normalized to the area. Scale bar = 0.5 μm. (D) Dual labeling of plasma membrane and GPER1 molecules defines the membrane regions (approximately 1 μm wide). Scale bar = 0.5 μm. (E) Line graph of the fluorescent intensity calculated from the magnified STED inserts (C2). (F) Integrated density graphs of GPER1 show the effect of vehicle and 100 nM of E2 treatment in the membrane and in the cytoplasm ( n = 15 cells were evaluated in each group). * p < 0.05.
    Figure Legend Snippet: The GluR2-AMPAR/GPER1 ratio and molecular distance between GPER1 and GluR2-AMPAR in the membrane. (A) STORM images depicting immunolabeled AMPAR (magenta) and GPER1 (cyan) molecules on dPC12. Dashed lines delineate the borders of the neurites and somas. Scale bar = 2 μm; inset Scale bar = 0.5 μm. (B) The ratio between the number of GPER1 and AMPAR molecules (GPER1/GluR2-AMPAR) on the neurites and somas ( n = 11 somas or neurites). (C1) Photomicrographs depict GPER1 immunoreactivity (visualized with STED microscopy) in dPC12 after 10 min of vehicle (left) or of 100 nM of E2 treatment (right). Scale bar = 2 μm. (C2) One 2 μm 2 (between parallel white bars) and one 10 μm 2 (to the left) areas were selected within each ROI for the membrane and cytoplasmic regions of each cell, respectively. Integrated density was calculated and normalized to the area. Scale bar = 0.5 μm. (D) Dual labeling of plasma membrane and GPER1 molecules defines the membrane regions (approximately 1 μm wide). Scale bar = 0.5 μm. (E) Line graph of the fluorescent intensity calculated from the magnified STED inserts (C2). (F) Integrated density graphs of GPER1 show the effect of vehicle and 100 nM of E2 treatment in the membrane and in the cytoplasm ( n = 15 cells were evaluated in each group). * p < 0.05.

    Techniques Used: Immunolabeling, Microscopy, Labeling

    The role of the cortical actin in the rapid effect of E2. (A) Left, confocal images depict Alexa Fluor 488 phalloidin-labeled cortical actin network in dPC12 after treatment with vehicle, 1 μM of latA, 1 μM of SP600125 or 1 μM of GSK429286. Scale bar = 5 μm; insert Scale bar = 0.5 μm. Right, the bar graph shows the effect of LatA, GSK429286, and SP600125 on the integrated density of the fluorescently labeled cortical actin network [ n = 3 cells per group (3 ROIs per cell)]. (B1,B2) Effect of LatA, GSK429286, and SP600125 treatment on D AMPAR (% of vehicle treatment as the mean ± SEM; n = 215–544 trajectories). (C1,C2) Effect of 100 pM of E2 on somas and 100 nM of E2 on neurites with or without LatA, GSK429286, and SP600125 (% of vehicle treatment as the mean ± SEM; n = 184–277 trajectories). *** p < 0.001.
    Figure Legend Snippet: The role of the cortical actin in the rapid effect of E2. (A) Left, confocal images depict Alexa Fluor 488 phalloidin-labeled cortical actin network in dPC12 after treatment with vehicle, 1 μM of latA, 1 μM of SP600125 or 1 μM of GSK429286. Scale bar = 5 μm; insert Scale bar = 0.5 μm. Right, the bar graph shows the effect of LatA, GSK429286, and SP600125 on the integrated density of the fluorescently labeled cortical actin network [ n = 3 cells per group (3 ROIs per cell)]. (B1,B2) Effect of LatA, GSK429286, and SP600125 treatment on D AMPAR (% of vehicle treatment as the mean ± SEM; n = 215–544 trajectories). (C1,C2) Effect of 100 pM of E2 on somas and 100 nM of E2 on neurites with or without LatA, GSK429286, and SP600125 (% of vehicle treatment as the mean ± SEM; n = 184–277 trajectories). *** p < 0.001.

    Techniques Used: Labeling

    Effect of E2 on the surface movement of GluR2-AMPA on primary hippocampal neurons. (A) Photomicrograph shows a primary hippocampal neuron labeled with homer-1 (synapse) and β-III tubulin (neuron). Scale bar = 10 μm, insert Scale bar = 2 μm. (B) Dual color STED image of a hippocampal neuron overlayed to differential interference contrast microscopy image depicts live-cell synapse labeling MitoTracker Deep Red (red) and presynaptic protein bassoon (green). Scale bar = 1 μm. (C) Distribution of D values of extrasynaptic and synaptic GluR2-AMPAR under control conditions (median ± IQR, n = 754 extrasynaptic trajectories and n = 104 synaptic trajectories). (D) Effect of E2 (100 pM and 100 nM) on D of extrasynaptic and synaptic GluR2-AMPA with or without chemical LTP (cLTP) induced by glycine/picrotoxin (gly/pic) (% of vehicle treatment as the mean ± SEM; n = 742–928 extrasynaptic trajectories and n = 104–155 synaptic trajectories). (E,F) Effect of vehicle, E2 (100 n, 100 pM) with or without cLTP (gly/pic) on synaptic dwell time (mean ± SEM (s); n = 104–155) (E) and relative surface distribution of synaptic GluR2-AMPAR content (synaptic/total GluR2-AMPA molecule trajectories) (mean ± SEM, n = 8–18 recordings) (F) . * p < 0.05; ** p < 0.01; *** p < 0.001.
    Figure Legend Snippet: Effect of E2 on the surface movement of GluR2-AMPA on primary hippocampal neurons. (A) Photomicrograph shows a primary hippocampal neuron labeled with homer-1 (synapse) and β-III tubulin (neuron). Scale bar = 10 μm, insert Scale bar = 2 μm. (B) Dual color STED image of a hippocampal neuron overlayed to differential interference contrast microscopy image depicts live-cell synapse labeling MitoTracker Deep Red (red) and presynaptic protein bassoon (green). Scale bar = 1 μm. (C) Distribution of D values of extrasynaptic and synaptic GluR2-AMPAR under control conditions (median ± IQR, n = 754 extrasynaptic trajectories and n = 104 synaptic trajectories). (D) Effect of E2 (100 pM and 100 nM) on D of extrasynaptic and synaptic GluR2-AMPA with or without chemical LTP (cLTP) induced by glycine/picrotoxin (gly/pic) (% of vehicle treatment as the mean ± SEM; n = 742–928 extrasynaptic trajectories and n = 104–155 synaptic trajectories). (E,F) Effect of vehicle, E2 (100 n, 100 pM) with or without cLTP (gly/pic) on synaptic dwell time (mean ± SEM (s); n = 104–155) (E) and relative surface distribution of synaptic GluR2-AMPAR content (synaptic/total GluR2-AMPA molecule trajectories) (mean ± SEM, n = 8–18 recordings) (F) . * p < 0.05; ** p < 0.01; *** p < 0.001.

    Techniques Used: Labeling, Microscopy

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    Alomone Labs glur2 ampar antibody
    Characterization of differentiated PC12 cells and validation of single-molecule labeling. (A,B) Left, Intensity profiles of a single ATTO 488-labeled <t>GluR2-AMPAR</t> (A) and mGluR1 (B) signal. The arrows indicate single-step photobleaching. Right, Histogram showing the intensity value of every spot found in a recording of ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) , superimposed with a single fitted lognormal curve (blue line). (C) Representative trajectories of AMPAR molecules on somas and neurites. Scale bar = 2 μm. (D) The mean square displacement functions and trajectories represent AMPAR molecules with Brownian motion (red) and confined motion (blue). Scale bar = 0.1 μm. (E,F) The cumulative probability functions of D values of AMPAR (E) and mGluR1 (F) on neurites and somas ( n = 510–676 trajectories). *** p < 0.001.
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    Image Search Results


    Validation of differentially expressed genes using qRT-PCR. ( A–E ) qPCR verification of the expression of genes involved in biological process identified as enriched by GO analysis compared with WT control. (F) Fold change expression of Ca 2+ -permeable AMPAR subunit Gria1 , Gria3 and Gria4 mRNAs, relative to WT motor neurons at E12.5. (G) Relative expression of Adarb1 mRNA in SOD1 G93A motor neurons at E12.5. ( H ) Schema showing the position of the fully complementary miR-124 target site at the 5′-end of the mouse Gria2 , 3′-UTR. The seed region of miR-124 is shown. Data represent mean ± SEM, unpaired student t -test, n = 5–7 biological replicates, * P < 0.05.

    Journal: Brain Communications

    Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

    doi: 10.1093/braincomms/fcac081

    Figure Lengend Snippet: Validation of differentially expressed genes using qRT-PCR. ( A–E ) qPCR verification of the expression of genes involved in biological process identified as enriched by GO analysis compared with WT control. (F) Fold change expression of Ca 2+ -permeable AMPAR subunit Gria1 , Gria3 and Gria4 mRNAs, relative to WT motor neurons at E12.5. (G) Relative expression of Adarb1 mRNA in SOD1 G93A motor neurons at E12.5. ( H ) Schema showing the position of the fully complementary miR-124 target site at the 5′-end of the mouse Gria2 , 3′-UTR. The seed region of miR-124 is shown. Data represent mean ± SEM, unpaired student t -test, n = 5–7 biological replicates, * P < 0.05.

    Article Snippet: Primary antibodies were as follows: chicken anti-GFP (1:1000; Abcam; AB13970), rabbit anti-NeuN (1:1000; Abcam; AB104225), goat anti-ChAT (1:500; Abcam; AB34419) and guinea pig anti-AMPA receptor 2 subunit (GluA2) (1:500; Alomone Labs; AGP-073).

    Techniques: Quantitative RT-PCR, Expressing

    Expression of GluA2 in spinal cords of embryonic SOD1 G93A mice. Cross-sections of lumbar spinal cord from WT (HB9:GFP; WT) and SOD1 G93A (SOD1 G93A ; HB9:GFP) mice at (A–J) E12.5 and ( K–T ) E17.5. Double-immunolabelling for GFP, GluA2 and NeuN (Neuronal nuclei). Plots represent quantification analysis of GluA2 signal intensity in HB9:GFP motor neurons at ( U ) E12.5 and ( V ) E17.5. Data represent mean ± SEM, unpaired student t -test performed on n = 4 biological replicates, ∼50 neurons analysed per biological replicate, * P < 0.05. Scale bars 50 μm.

    Journal: Brain Communications

    Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

    doi: 10.1093/braincomms/fcac081

    Figure Lengend Snippet: Expression of GluA2 in spinal cords of embryonic SOD1 G93A mice. Cross-sections of lumbar spinal cord from WT (HB9:GFP; WT) and SOD1 G93A (SOD1 G93A ; HB9:GFP) mice at (A–J) E12.5 and ( K–T ) E17.5. Double-immunolabelling for GFP, GluA2 and NeuN (Neuronal nuclei). Plots represent quantification analysis of GluA2 signal intensity in HB9:GFP motor neurons at ( U ) E12.5 and ( V ) E17.5. Data represent mean ± SEM, unpaired student t -test performed on n = 4 biological replicates, ∼50 neurons analysed per biological replicate, * P < 0.05. Scale bars 50 μm.

    Article Snippet: Primary antibodies were as follows: chicken anti-GFP (1:1000; Abcam; AB13970), rabbit anti-NeuN (1:1000; Abcam; AB104225), goat anti-ChAT (1:500; Abcam; AB34419) and guinea pig anti-AMPA receptor 2 subunit (GluA2) (1:500; Alomone Labs; AGP-073).

    Techniques: Expressing

    Expression of GRIA2 and ADAR2 in iPSC motor neurons derived from ALS patients with SOD1 mutations and healthy control lines. Representative images of iPSC mature motor neurons derived from ( A–E ) healthy control line and ( F–J ) SOD1 I114T line, immunolabelled with ChAT, GluA2 and TUJ1, counterstained with Hoechst. ( K ) Plot represents quantification analysis of GluA2 signal intensity in iPSC motor neurons. Data represent mean ± SEM, unpaired student t -test performed on n = 3 biological replicates, 50 neurons analysed per biological replicate. (L) Fold change expression of GRIA2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. ( M ) Fold change expression of ADAR2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. Data represent mean ± SEM, n = 3 biological replicates, one-way ANOVA with Dunnett's multiple comparison test, * P < 0.01, ** P < 0.005. Scale bars 50 μm.

    Journal: Brain Communications

    Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

    doi: 10.1093/braincomms/fcac081

    Figure Lengend Snippet: Expression of GRIA2 and ADAR2 in iPSC motor neurons derived from ALS patients with SOD1 mutations and healthy control lines. Representative images of iPSC mature motor neurons derived from ( A–E ) healthy control line and ( F–J ) SOD1 I114T line, immunolabelled with ChAT, GluA2 and TUJ1, counterstained with Hoechst. ( K ) Plot represents quantification analysis of GluA2 signal intensity in iPSC motor neurons. Data represent mean ± SEM, unpaired student t -test performed on n = 3 biological replicates, 50 neurons analysed per biological replicate. (L) Fold change expression of GRIA2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. ( M ) Fold change expression of ADAR2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. Data represent mean ± SEM, n = 3 biological replicates, one-way ANOVA with Dunnett's multiple comparison test, * P < 0.01, ** P < 0.005. Scale bars 50 μm.

    Article Snippet: Primary antibodies were as follows: chicken anti-GFP (1:1000; Abcam; AB13970), rabbit anti-NeuN (1:1000; Abcam; AB104225), goat anti-ChAT (1:500; Abcam; AB34419) and guinea pig anti-AMPA receptor 2 subunit (GluA2) (1:500; Alomone Labs; AGP-073).

    Techniques: Expressing, Derivative Assay, Quantitative RT-PCR

    Validation of differentially expressed genes using qRT-PCR. ( A–E ) qPCR verification of the expression of genes involved in biological process identified as enriched by GO analysis compared with WT control. (F) Fold change expression of Ca 2+ -permeable AMPAR subunit Gria1 , Gria3 and Gria4 mRNAs, relative to WT motor neurons at E12.5. (G) Relative expression of Adarb1 mRNA in SOD1 G93A motor neurons at E12.5. ( H ) Schema showing the position of the fully complementary miR-124 target site at the 5′-end of the mouse Gria2 , 3′-UTR. The seed region of miR-124 is shown. Data represent mean ± SEM, unpaired student t -test, n = 5–7 biological replicates, * P < 0.05.

    Journal: Brain Communications

    Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

    doi: 10.1093/braincomms/fcac081

    Figure Lengend Snippet: Validation of differentially expressed genes using qRT-PCR. ( A–E ) qPCR verification of the expression of genes involved in biological process identified as enriched by GO analysis compared with WT control. (F) Fold change expression of Ca 2+ -permeable AMPAR subunit Gria1 , Gria3 and Gria4 mRNAs, relative to WT motor neurons at E12.5. (G) Relative expression of Adarb1 mRNA in SOD1 G93A motor neurons at E12.5. ( H ) Schema showing the position of the fully complementary miR-124 target site at the 5′-end of the mouse Gria2 , 3′-UTR. The seed region of miR-124 is shown. Data represent mean ± SEM, unpaired student t -test, n = 5–7 biological replicates, * P < 0.05.

    Article Snippet: Cells were blocked in 10% (v/v) normal donkey serum with 0.1% (v/v) Triton-X 100 in 0.1 M PBS and incubated overnight at 4°C in the following primary antibodies: goat anti-ChAT (1:500, Millipore; AB144P), chicken anti-β III tubulin (1:1000, Abcam; AB41489) and rabbit anti-GluA2 (1:500; Alomone Labs; AGC-005).

    Techniques: Quantitative RT-PCR, Expressing

    Expression of GluA2 in spinal cords of embryonic SOD1 G93A mice. Cross-sections of lumbar spinal cord from WT (HB9:GFP; WT) and SOD1 G93A (SOD1 G93A ; HB9:GFP) mice at (A–J) E12.5 and ( K–T ) E17.5. Double-immunolabelling for GFP, GluA2 and NeuN (Neuronal nuclei). Plots represent quantification analysis of GluA2 signal intensity in HB9:GFP motor neurons at ( U ) E12.5 and ( V ) E17.5. Data represent mean ± SEM, unpaired student t -test performed on n = 4 biological replicates, ∼50 neurons analysed per biological replicate, * P < 0.05. Scale bars 50 μm.

    Journal: Brain Communications

    Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

    doi: 10.1093/braincomms/fcac081

    Figure Lengend Snippet: Expression of GluA2 in spinal cords of embryonic SOD1 G93A mice. Cross-sections of lumbar spinal cord from WT (HB9:GFP; WT) and SOD1 G93A (SOD1 G93A ; HB9:GFP) mice at (A–J) E12.5 and ( K–T ) E17.5. Double-immunolabelling for GFP, GluA2 and NeuN (Neuronal nuclei). Plots represent quantification analysis of GluA2 signal intensity in HB9:GFP motor neurons at ( U ) E12.5 and ( V ) E17.5. Data represent mean ± SEM, unpaired student t -test performed on n = 4 biological replicates, ∼50 neurons analysed per biological replicate, * P < 0.05. Scale bars 50 μm.

    Article Snippet: Cells were blocked in 10% (v/v) normal donkey serum with 0.1% (v/v) Triton-X 100 in 0.1 M PBS and incubated overnight at 4°C in the following primary antibodies: goat anti-ChAT (1:500, Millipore; AB144P), chicken anti-β III tubulin (1:1000, Abcam; AB41489) and rabbit anti-GluA2 (1:500; Alomone Labs; AGC-005).

    Techniques: Expressing

    Expression of GRIA2 and ADAR2 in iPSC motor neurons derived from ALS patients with SOD1 mutations and healthy control lines. Representative images of iPSC mature motor neurons derived from ( A–E ) healthy control line and ( F–J ) SOD1 I114T line, immunolabelled with ChAT, GluA2 and TUJ1, counterstained with Hoechst. ( K ) Plot represents quantification analysis of GluA2 signal intensity in iPSC motor neurons. Data represent mean ± SEM, unpaired student t -test performed on n = 3 biological replicates, 50 neurons analysed per biological replicate. (L) Fold change expression of GRIA2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. ( M ) Fold change expression of ADAR2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. Data represent mean ± SEM, n = 3 biological replicates, one-way ANOVA with Dunnett's multiple comparison test, * P < 0.01, ** P < 0.005. Scale bars 50 μm.

    Journal: Brain Communications

    Article Title: α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

    doi: 10.1093/braincomms/fcac081

    Figure Lengend Snippet: Expression of GRIA2 and ADAR2 in iPSC motor neurons derived from ALS patients with SOD1 mutations and healthy control lines. Representative images of iPSC mature motor neurons derived from ( A–E ) healthy control line and ( F–J ) SOD1 I114T line, immunolabelled with ChAT, GluA2 and TUJ1, counterstained with Hoechst. ( K ) Plot represents quantification analysis of GluA2 signal intensity in iPSC motor neurons. Data represent mean ± SEM, unpaired student t -test performed on n = 3 biological replicates, 50 neurons analysed per biological replicate. (L) Fold change expression of GRIA2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. ( M ) Fold change expression of ADAR2 in SOD1 lines, compared with healthy control line determined by qRT-PCR. Data represent mean ± SEM, n = 3 biological replicates, one-way ANOVA with Dunnett's multiple comparison test, * P < 0.01, ** P < 0.005. Scale bars 50 μm.

    Article Snippet: Cells were blocked in 10% (v/v) normal donkey serum with 0.1% (v/v) Triton-X 100 in 0.1 M PBS and incubated overnight at 4°C in the following primary antibodies: goat anti-ChAT (1:500, Millipore; AB144P), chicken anti-β III tubulin (1:1000, Abcam; AB41489) and rabbit anti-GluA2 (1:500; Alomone Labs; AGC-005).

    Techniques: Expressing, Derivative Assay, Quantitative RT-PCR

    Astaxanthin inhibits a [Ca 2+ ]i increase in cortical neurons upon ionotropic glutamate receptor activation. ( A – C ) The average [Ca 2+ ]i response in control (black) and AST (red) preincubated neurons stimulated with 50 μM each of NMDA (+ 5 μM glycine), AMPA and KA for 15 min (NMDA: Con n = 23, AST n = 42; AMPA: con n = 27, AST n = 23; KA con n = 30, AST n = 40). ( D ) Dot plot representing the total calcium (area under the curve) after 15 min of NMDA, AMPA and KA stimulation. Arrow heads indicate point of glutamate receptor agonist applications. ( E ) Representative protein expression levels of NMDA (GluN1), AMPA (GluA2) and KA (GluK123) detected by the Western blot analysis with β-actin as the internal reference (individual Western blots figure are provided in ). ( F ) Dot plot indicate the average normalized protein expression for GluN1, GluA2 and GluK123. Data are represented as mean ± SEM from 3–4 different experiments, * p < 0.05. n.s: non-significant.

    Journal: Marine Drugs

    Article Title: Astaxanthin Protection against Neuronal Excitotoxicity via Glutamate Receptor Inhibition and Improvement of Mitochondrial Function

    doi: 10.3390/md20100645

    Figure Lengend Snippet: Astaxanthin inhibits a [Ca 2+ ]i increase in cortical neurons upon ionotropic glutamate receptor activation. ( A – C ) The average [Ca 2+ ]i response in control (black) and AST (red) preincubated neurons stimulated with 50 μM each of NMDA (+ 5 μM glycine), AMPA and KA for 15 min (NMDA: Con n = 23, AST n = 42; AMPA: con n = 27, AST n = 23; KA con n = 30, AST n = 40). ( D ) Dot plot representing the total calcium (area under the curve) after 15 min of NMDA, AMPA and KA stimulation. Arrow heads indicate point of glutamate receptor agonist applications. ( E ) Representative protein expression levels of NMDA (GluN1), AMPA (GluA2) and KA (GluK123) detected by the Western blot analysis with β-actin as the internal reference (individual Western blots figure are provided in ). ( F ) Dot plot indicate the average normalized protein expression for GluN1, GluA2 and GluK123. Data are represented as mean ± SEM from 3–4 different experiments, * p < 0.05. n.s: non-significant.

    Article Snippet: Membranes were then probed with specific primary antibodies GluN1 (NMDAR1, 1:1000, Alomone Lab Jerusalem, Israel), GluR2 (GluA2, 1:1000, Alomone Lab), GluK 123 (GluR 567, 1:2000, Santa Cruz, Dallas, TX, USA) and β-Actin, 1:2000, BD).

    Techniques: Activation Assay, Expressing, Western Blot

    a Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled for surface GluA2 (under non-permeabilizing conditions). Graph showing the number of surface GluA2 labeling, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA, F (2, 15) = 38.28) (Tukey’s test P control-NMDAR < 0.0001, P control-mGluR < 0.0001, P NMDAR-mGluR = 0.8438). b Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled with an antibody against endogenous LC3 (autophagic structures) and MAP2 (dendrites). Graph showing the number of dendritic LC3-positive puncta in secondary dendrites, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F2,24) = 15.11, P < 0.0001) (Tukey’s test Pcontrol-NMDA = 0.0005, P control-mGluR = 0.0001). c Same as in b , but neurons were pretreated for 1 h before, during and after the pulse with wortmannin (500 nM) or SBI-0206965 (500 nM). Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions (U: untreated, W: wortmannin, S: SBI-0206965). Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(8,45) = 33.83, P < 0.0001) (Tukey’s test P control/S-NMDA/S = 0.3677, P control/W-NMDA/W = 0.9986, P NMDA/U-NMDA/W < 0.0001, P NMDA/U-NMDA/S < 0.0001, P control/S-DHPG/S = 0.9674, P control/W-DHPG/W = 0.9989, P DHPG/U-DHPG/W < 0.0001, P DHPG/U-DHPG/S < 0.0001). d Same as in b with neurons that were infected with AAV plasmids carrying 4 shRNA sequences against atg5 ( sh-atg5 ) or scrambled control ( sh-scramble ), under the CamK2a promoter. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(5,30) = 16.94, P < 0.0001) (Tukey’s test P control/scr-control/atg5 = 0.9999, P NMDA/scr-NMDA/atg5 = 0.0025, P DHPG/scr-DHPG/atg5 < 0.0001, P control/scr-NMDA/scr < 0.0001, P control/scr-DGPG/scr < 0.0001, P control/atg5-NMDA/atg5 = 0.8959, P control/atg5-DHPG/atg5 = 0.9637). e Same as in b , but neurons were immunolabeled 15 min after NMDAR- and mGluR-LTD and treated for 1 h before, during and after the pulse with Ifenprodil (10 μM) or MTEP (10 μM) and JNJ16259685 (10 μM) to pharmacologically inhibit NR2B and mGluR1/5 receptors, respectively. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F (3,32) = 74.46, P < 0.0001) (Tukey’s test, P NMDA-NMDA+IFE < 0.0001, P DHPG-DHPG+MTEP/JNJ < 0.0001). Scale bars: 10 μm for all panels.

    Journal: Nature Communications

    Article Title: Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice

    doi: 10.1038/s41467-022-28301-z

    Figure Lengend Snippet: a Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled for surface GluA2 (under non-permeabilizing conditions). Graph showing the number of surface GluA2 labeling, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA, F (2, 15) = 38.28) (Tukey’s test P control-NMDAR < 0.0001, P control-mGluR < 0.0001, P NMDAR-mGluR = 0.8438). b Representative confocal images of cultured neurons under control conditions or 15 min after chemical NMDAR- or mGluR-LTD, immunolabeled with an antibody against endogenous LC3 (autophagic structures) and MAP2 (dendrites). Graph showing the number of dendritic LC3-positive puncta in secondary dendrites, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F2,24) = 15.11, P < 0.0001) (Tukey’s test Pcontrol-NMDA = 0.0005, P control-mGluR = 0.0001). c Same as in b , but neurons were pretreated for 1 h before, during and after the pulse with wortmannin (500 nM) or SBI-0206965 (500 nM). Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions (U: untreated, W: wortmannin, S: SBI-0206965). Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(8,45) = 33.83, P < 0.0001) (Tukey’s test P control/S-NMDA/S = 0.3677, P control/W-NMDA/W = 0.9986, P NMDA/U-NMDA/W < 0.0001, P NMDA/U-NMDA/S < 0.0001, P control/S-DHPG/S = 0.9674, P control/W-DHPG/W = 0.9989, P DHPG/U-DHPG/W < 0.0001, P DHPG/U-DHPG/S < 0.0001). d Same as in b with neurons that were infected with AAV plasmids carrying 4 shRNA sequences against atg5 ( sh-atg5 ) or scrambled control ( sh-scramble ), under the CamK2a promoter. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. Bars represent mean values ± SEM. N = 6 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F(5,30) = 16.94, P < 0.0001) (Tukey’s test P control/scr-control/atg5 = 0.9999, P NMDA/scr-NMDA/atg5 = 0.0025, P DHPG/scr-DHPG/atg5 < 0.0001, P control/scr-NMDA/scr < 0.0001, P control/scr-DGPG/scr < 0.0001, P control/atg5-NMDA/atg5 = 0.8959, P control/atg5-DHPG/atg5 = 0.9637). e Same as in b , but neurons were immunolabeled 15 min after NMDAR- and mGluR-LTD and treated for 1 h before, during and after the pulse with Ifenprodil (10 μM) or MTEP (10 μM) and JNJ16259685 (10 μM) to pharmacologically inhibit NR2B and mGluR1/5 receptors, respectively. Graph showing the number of dendritic LC3-positive puncta, normalized to the dendritic length, in the indicated conditions. N = 9 independent experiments per condition. Statistical analyses were performed by one-way ANOVA (F (3,32) = 74.46, P < 0.0001) (Tukey’s test, P NMDA-NMDA+IFE < 0.0001, P DHPG-DHPG+MTEP/JNJ < 0.0001). Scale bars: 10 μm for all panels.

    Article Snippet: The primary antibodies used were: LC3 (1:1000, Santa Cruz, sc-376404 and Sigma, L7543), Atg13 (1:1000, Sigma–Aldrich, SAB4200100), MAP2 (1:1000, Synaptic Systems, #188004), FIP200 (1:1000, Cell Signaling, #12436), Atg101 (1:1000, Cell Signaling, #13492), ULK1 (1:1000, Cell Signaling, #8054), p62 (1:2000, Calbiochem, DR1057), WIPI2 (1:1000, Abcam, ab105459), GluA2 (N-terminus, 1:1000, Alomone, AGP-073), PSD95 (1:1000, Invitrogen, MA1-046) and Arc (1:1000, Synaptic Systems, #156003).

    Techniques: Cell Culture, Immunolabeling, Labeling, Infection, shRNA

    a Confocal images of dendrites immunolabeled with an antibody against the extracellular region of GluA2 under control conditions or 15 min after LTD induction and in the absence or presence of Dynamin-1 inhibitory peptide (50 µM) or SBI-0206965 (500 nM), a selective inhibitor of the ULK1 kinase activity. Inhibitors were applied 25 min before, during and 15 min after the pulses. Scale bar: 10 µm. Graph showing the surface labeling of GluA2, normalized to dendritic length under the aforementioned conditions. Bars represent mean values ± SEM. N = 9 independent experiments. Statistical analysis was performed using one-way ANOVA (F (8, 72) = 7.411, P < 0.0001) (Tukey’s test P control-control/D > 0.99, P control-control/S = 0.9971, P NMDA-NMDA/D = 0.0451, P NMDA-NMDA/S = 0.0008, P DHPG-DHPG/D = 0.0017, P DHPG-DHPG/S = 0.0002). b Confocal images of dendrites of neurons expressing 4 scrambled sequences ( sh-scramble ), or 4 sh-RNAs against atg5 ( sh-atg5 ), immunolabeled with an antibody against the extracellular region of GluA2 under control conditions or 15 min after LTD induction. Graph showing the surface labeling of GluA2, normalized to dendritic length under the aforementioned conditions. Bars represent mean values ± SEM. N = 10 independent experiments. Statistical analysis was performed using one-way ANOVA (F (5, 54) = 30.02, P < 0.0001) (Tukey’s test, P control/scr-control/atg5 = 0.0626, P NMDA/scr-NMDA/atg5 < 0.0001, P DHPG/scr-DHPG/atg5 < 0.0001, P control/atg5-NMDA/atg5 > 0.99, P control/atg5-DHPG/atg5 = 0.8602, P control/scr-NMDA/scr = 0.0008, P control/scr-DHPG/scr < 0.0001). c Representative images of consecutive confocal z-planes of cultured neurons immunostained with antibodies against PSD95, LC3, and MAP2 to label the dendrites, 15 min after cLTD. Note the colocalization of PSD95 and LC3 in dendritic spines (yellow arrows) and in the dendritic shaft (white arrows), in consecutive z-planes. Scale bar: 10 µm. Graph showing the percentage of PSD95 puncta co-localizing with LC3 in consecutive confocal z-planes in dendritic spines and shafts in control neurons or 15 min after chemically induced NMDAR- or mGluR-LTD. Bars represent mean values ± SEM. N = 8 independent experiments. Statistical analysis was performed by one-way ANOVA (F(5,42) = 48.43, P < 0.0001) (Tukey’s test for dendritic shaft, P control-NMDA = 0.0569, P control-DHPG = 0.1948, for dendritic spines, P control-NMDA < 0.0001, P control-DHPG < 0.0001). d Western blot analysis for GluA2 and PSD95 in lysates of cultured neurons in control conditions or 15 min after NMDAR- and mGluR-LTD and in the presence or absence of Bafilomycin A1 (50 µM) for 15 min before, during, and 15 min after the NMDA and DHPG pulses. e Western blot analysis for GluA2 and PSD95 in lysates of cultured neurons in control conditions or 15 min after NMDAR- and mGluR-LTD and in the presence or absence of SBI-0206965 (500 nM) for 30 min before, during, and 15 min after the NMDA and DHPG pulses. f Western blot analysis for GluA2 and PSD95 in lysates of cultured shscrambled or sh-atg5 expressing neurons in control conditions or 15 min after NMDAR- and mGluR-LTD. d – f Graphs showing the levels of PSD95 and GluA2 levels in the indicated conditions, normalized to total protein levels. Bars represent mean values ± SEM. Statistical analysis was performed by one-way ANOVA. d (N = 9 independent experiments) PSD95: F(5,48) = 15.08, P < 0.0001 (Tukey’s test P control-control/Baf = 0.7566, P control-NMDA = 0.0016, P control-DHPG = 0.0081, P NMDA-NMDA/Baf < 0.0001, P DHPG-DHPG/Baf = 0.0013. GluA2: F(5,48)=6.627, P < 0.0001 (Tukey’s test P control-control/Baf = 0.9692, P control-NMDA = 0.0014, P control-DHPG = 0.0067, P NMDA-NMDA/Baf = 0.0421, P DHPG-DHPG/Baf = 0.0127. e ( N = 7 independent experiments) PSD95: F(5,36) = 23.80, P < 0.0001. (Tukey’s test P control-control/SBI > 0.99, P NMDA-NMDA/SBI < 0.0001, P DHPG-DHPG/SBI < 0.0001, P control-NMDA < 0.0001, P control-DHPG < 0.0001, P control/SBI-NMDA/SBI = 0.9764, P control/SBI-DHPG/SBI = 0.6286). Panel e, GluA2: F(5,36)=11.73, P < 0.0001. (Tukey’s test P control-control/SBI = 0.9179, P NMDA-NMDA/SBI = 0.0001, P DHPG-DHPG/SBI = 0.0002, P control-NMDA = 0.0099, P control-DHPG = 0.0323, P control/SBI-NMDA/SBI = 0.9959, P control/SBI-DHPG/SBI = 0.9407). f ( N = 7 independent experiments) PSD95: F(5,36) = 10.93, P < 0.0001. (Tukey’s test P control/scr-control/atg5 = 0.7927, P NMDA/scr-NMDA/atg5 = 0.0045, P DHPG/scr-DHPG/atg5 = 0.0003, P control/scr-NMDA/scr = 0.0134, P control/scr-DHPG/scr = 0.0030, P control/atg5-NMDA/atg5 = 0.9488, P control/atg5-DHPG/atg5 = 0.9976). GluA2: F(5,36) = 10.79, P < 0.0001. (Tukey’s test P control/scr-control/atg5 > 0.99, P NMDA/scr-NMDA/atg5 = 0,0001, P DHPG/scr-DHPG/atg5 = 0.0019, P control/scr-NMDA/scr = 0.0134, P control/scr-DHPG/scr = 0.0021, P control/atg5-NMDA/atg5 = 0.5844, P control/atg5-DHPG/atg5 > 0.99).

    Journal: Nature Communications

    Article Title: Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice

    doi: 10.1038/s41467-022-28301-z

    Figure Lengend Snippet: a Confocal images of dendrites immunolabeled with an antibody against the extracellular region of GluA2 under control conditions or 15 min after LTD induction and in the absence or presence of Dynamin-1 inhibitory peptide (50 µM) or SBI-0206965 (500 nM), a selective inhibitor of the ULK1 kinase activity. Inhibitors were applied 25 min before, during and 15 min after the pulses. Scale bar: 10 µm. Graph showing the surface labeling of GluA2, normalized to dendritic length under the aforementioned conditions. Bars represent mean values ± SEM. N = 9 independent experiments. Statistical analysis was performed using one-way ANOVA (F (8, 72) = 7.411, P < 0.0001) (Tukey’s test P control-control/D > 0.99, P control-control/S = 0.9971, P NMDA-NMDA/D = 0.0451, P NMDA-NMDA/S = 0.0008, P DHPG-DHPG/D = 0.0017, P DHPG-DHPG/S = 0.0002). b Confocal images of dendrites of neurons expressing 4 scrambled sequences ( sh-scramble ), or 4 sh-RNAs against atg5 ( sh-atg5 ), immunolabeled with an antibody against the extracellular region of GluA2 under control conditions or 15 min after LTD induction. Graph showing the surface labeling of GluA2, normalized to dendritic length under the aforementioned conditions. Bars represent mean values ± SEM. N = 10 independent experiments. Statistical analysis was performed using one-way ANOVA (F (5, 54) = 30.02, P < 0.0001) (Tukey’s test, P control/scr-control/atg5 = 0.0626, P NMDA/scr-NMDA/atg5 < 0.0001, P DHPG/scr-DHPG/atg5 < 0.0001, P control/atg5-NMDA/atg5 > 0.99, P control/atg5-DHPG/atg5 = 0.8602, P control/scr-NMDA/scr = 0.0008, P control/scr-DHPG/scr < 0.0001). c Representative images of consecutive confocal z-planes of cultured neurons immunostained with antibodies against PSD95, LC3, and MAP2 to label the dendrites, 15 min after cLTD. Note the colocalization of PSD95 and LC3 in dendritic spines (yellow arrows) and in the dendritic shaft (white arrows), in consecutive z-planes. Scale bar: 10 µm. Graph showing the percentage of PSD95 puncta co-localizing with LC3 in consecutive confocal z-planes in dendritic spines and shafts in control neurons or 15 min after chemically induced NMDAR- or mGluR-LTD. Bars represent mean values ± SEM. N = 8 independent experiments. Statistical analysis was performed by one-way ANOVA (F(5,42) = 48.43, P < 0.0001) (Tukey’s test for dendritic shaft, P control-NMDA = 0.0569, P control-DHPG = 0.1948, for dendritic spines, P control-NMDA < 0.0001, P control-DHPG < 0.0001). d Western blot analysis for GluA2 and PSD95 in lysates of cultured neurons in control conditions or 15 min after NMDAR- and mGluR-LTD and in the presence or absence of Bafilomycin A1 (50 µM) for 15 min before, during, and 15 min after the NMDA and DHPG pulses. e Western blot analysis for GluA2 and PSD95 in lysates of cultured neurons in control conditions or 15 min after NMDAR- and mGluR-LTD and in the presence or absence of SBI-0206965 (500 nM) for 30 min before, during, and 15 min after the NMDA and DHPG pulses. f Western blot analysis for GluA2 and PSD95 in lysates of cultured shscrambled or sh-atg5 expressing neurons in control conditions or 15 min after NMDAR- and mGluR-LTD. d – f Graphs showing the levels of PSD95 and GluA2 levels in the indicated conditions, normalized to total protein levels. Bars represent mean values ± SEM. Statistical analysis was performed by one-way ANOVA. d (N = 9 independent experiments) PSD95: F(5,48) = 15.08, P < 0.0001 (Tukey’s test P control-control/Baf = 0.7566, P control-NMDA = 0.0016, P control-DHPG = 0.0081, P NMDA-NMDA/Baf < 0.0001, P DHPG-DHPG/Baf = 0.0013. GluA2: F(5,48)=6.627, P < 0.0001 (Tukey’s test P control-control/Baf = 0.9692, P control-NMDA = 0.0014, P control-DHPG = 0.0067, P NMDA-NMDA/Baf = 0.0421, P DHPG-DHPG/Baf = 0.0127. e ( N = 7 independent experiments) PSD95: F(5,36) = 23.80, P < 0.0001. (Tukey’s test P control-control/SBI > 0.99, P NMDA-NMDA/SBI < 0.0001, P DHPG-DHPG/SBI < 0.0001, P control-NMDA < 0.0001, P control-DHPG < 0.0001, P control/SBI-NMDA/SBI = 0.9764, P control/SBI-DHPG/SBI = 0.6286). Panel e, GluA2: F(5,36)=11.73, P < 0.0001. (Tukey’s test P control-control/SBI = 0.9179, P NMDA-NMDA/SBI = 0.0001, P DHPG-DHPG/SBI = 0.0002, P control-NMDA = 0.0099, P control-DHPG = 0.0323, P control/SBI-NMDA/SBI = 0.9959, P control/SBI-DHPG/SBI = 0.9407). f ( N = 7 independent experiments) PSD95: F(5,36) = 10.93, P < 0.0001. (Tukey’s test P control/scr-control/atg5 = 0.7927, P NMDA/scr-NMDA/atg5 = 0.0045, P DHPG/scr-DHPG/atg5 = 0.0003, P control/scr-NMDA/scr = 0.0134, P control/scr-DHPG/scr = 0.0030, P control/atg5-NMDA/atg5 = 0.9488, P control/atg5-DHPG/atg5 = 0.9976). GluA2: F(5,36) = 10.79, P < 0.0001. (Tukey’s test P control/scr-control/atg5 > 0.99, P NMDA/scr-NMDA/atg5 = 0,0001, P DHPG/scr-DHPG/atg5 = 0.0019, P control/scr-NMDA/scr = 0.0134, P control/scr-DHPG/scr = 0.0021, P control/atg5-NMDA/atg5 = 0.5844, P control/atg5-DHPG/atg5 > 0.99).

    Article Snippet: The primary antibodies used were: LC3 (1:1000, Santa Cruz, sc-376404 and Sigma, L7543), Atg13 (1:1000, Sigma–Aldrich, SAB4200100), MAP2 (1:1000, Synaptic Systems, #188004), FIP200 (1:1000, Cell Signaling, #12436), Atg101 (1:1000, Cell Signaling, #13492), ULK1 (1:1000, Cell Signaling, #8054), p62 (1:2000, Calbiochem, DR1057), WIPI2 (1:1000, Abcam, ab105459), GluA2 (N-terminus, 1:1000, Alomone, AGP-073), PSD95 (1:1000, Invitrogen, MA1-046) and Arc (1:1000, Synaptic Systems, #156003).

    Techniques: Immunolabeling, Activity Assay, Labeling, Expressing, Cell Culture, Western Blot

    a – d Western blot analyses of different fractions along the autophagic vesicle purification procedure, using antibodies against a autophagosomal markers (LC3B, p62, Atg16L1, and Atg9A), b ER-Golgi markers (TGN, LMAN1, SAR1a), c endosomal markers (Rab11b, EEA1), and d markers of the plasma-membrane (Stx4), extracellular vesicles (Alix) and nuclear extracts (TBP). N = 3 independent experiments. e Graph showing the cell component analysis, as false discovery rate (FDR)-corrected p -values, of the dynamic cargo (total of 393 proteins) that is enriched (up) or less abundant (down) in AVs after LTD, compared to control. f Graphical representation of proteins enriched in AVs upon LTD, with relation to the synapse. g Western blot analysis of PK-treated control and LTD-AVs, validating the presence of the proteins identified by the proteomic analyses in the autophagic vesicles. Postsynaptic density (PSD) fraction was used as reference. Graph showing the fold change of the normalized levels of the proteins validated by western blot, as a ratio of LTD to control. Cargo proteins were normalized to the levels of p62, which remains unaffected at the early phase of LTD. N = 3 independent AV preparations. Bars represent mean values ± SEM. Statistical analysis was performed using paired, two-tailed Student’s t -test (GluA1, N = 6, P = 0.0002; GluA2, N = 6, P = 0.0039; Pick1, N = 5, P = 0.011; SAP97, N = 5, P = 0.0179; FYN, N = 8, P < 0.0001; CamKIIa, N = 8, P < 0.0001; IL1RAPL1, N = 8, P = 0.0004; Adam22, N = 4, P = 0.0018; INA, N = 3, P = 0.0287; MYH10, N = 8, P < 0.0001; ITPKA, N = 6, P = 0.0006; KCC2, N = 4, P = 0. 0352; cofilin-1, N = 6, P = 0.005; dynamin, N = 6, P = 0.0005; p62, N = 6, P = 0.9809). All indicated molecular weights in a – d and g are in kDaltons (kD).

    Journal: Nature Communications

    Article Title: Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice

    doi: 10.1038/s41467-022-28301-z

    Figure Lengend Snippet: a – d Western blot analyses of different fractions along the autophagic vesicle purification procedure, using antibodies against a autophagosomal markers (LC3B, p62, Atg16L1, and Atg9A), b ER-Golgi markers (TGN, LMAN1, SAR1a), c endosomal markers (Rab11b, EEA1), and d markers of the plasma-membrane (Stx4), extracellular vesicles (Alix) and nuclear extracts (TBP). N = 3 independent experiments. e Graph showing the cell component analysis, as false discovery rate (FDR)-corrected p -values, of the dynamic cargo (total of 393 proteins) that is enriched (up) or less abundant (down) in AVs after LTD, compared to control. f Graphical representation of proteins enriched in AVs upon LTD, with relation to the synapse. g Western blot analysis of PK-treated control and LTD-AVs, validating the presence of the proteins identified by the proteomic analyses in the autophagic vesicles. Postsynaptic density (PSD) fraction was used as reference. Graph showing the fold change of the normalized levels of the proteins validated by western blot, as a ratio of LTD to control. Cargo proteins were normalized to the levels of p62, which remains unaffected at the early phase of LTD. N = 3 independent AV preparations. Bars represent mean values ± SEM. Statistical analysis was performed using paired, two-tailed Student’s t -test (GluA1, N = 6, P = 0.0002; GluA2, N = 6, P = 0.0039; Pick1, N = 5, P = 0.011; SAP97, N = 5, P = 0.0179; FYN, N = 8, P < 0.0001; CamKIIa, N = 8, P < 0.0001; IL1RAPL1, N = 8, P = 0.0004; Adam22, N = 4, P = 0.0018; INA, N = 3, P = 0.0287; MYH10, N = 8, P < 0.0001; ITPKA, N = 6, P = 0.0006; KCC2, N = 4, P = 0. 0352; cofilin-1, N = 6, P = 0.005; dynamin, N = 6, P = 0.0005; p62, N = 6, P = 0.9809). All indicated molecular weights in a – d and g are in kDaltons (kD).

    Article Snippet: The primary antibodies used were: LC3 (1:1000, Santa Cruz, sc-376404 and Sigma, L7543), Atg13 (1:1000, Sigma–Aldrich, SAB4200100), MAP2 (1:1000, Synaptic Systems, #188004), FIP200 (1:1000, Cell Signaling, #12436), Atg101 (1:1000, Cell Signaling, #13492), ULK1 (1:1000, Cell Signaling, #8054), p62 (1:2000, Calbiochem, DR1057), WIPI2 (1:1000, Abcam, ab105459), GluA2 (N-terminus, 1:1000, Alomone, AGP-073), PSD95 (1:1000, Invitrogen, MA1-046) and Arc (1:1000, Synaptic Systems, #156003).

    Techniques: Western Blot, Purification, Two Tailed Test

    Journal: Nature Communications

    Article Title: Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice

    doi: 10.1038/s41467-022-28301-z

    Figure Lengend Snippet:

    Article Snippet: The primary antibodies used were: LC3 (1:1000, Santa Cruz, sc-376404 and Sigma, L7543), Atg13 (1:1000, Sigma–Aldrich, SAB4200100), MAP2 (1:1000, Synaptic Systems, #188004), FIP200 (1:1000, Cell Signaling, #12436), Atg101 (1:1000, Cell Signaling, #13492), ULK1 (1:1000, Cell Signaling, #8054), p62 (1:2000, Calbiochem, DR1057), WIPI2 (1:1000, Abcam, ab105459), GluA2 (N-terminus, 1:1000, Alomone, AGP-073), PSD95 (1:1000, Invitrogen, MA1-046) and Arc (1:1000, Synaptic Systems, #156003).

    Techniques: Concentration Assay, Activity Assay, Plasmid Preparation, Avidin-Biotin Assay, Infection, In Vivo

    Characterization of differentiated PC12 cells and validation of single-molecule labeling. (A,B) Left, Intensity profiles of a single ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) signal. The arrows indicate single-step photobleaching. Right, Histogram showing the intensity value of every spot found in a recording of ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) , superimposed with a single fitted lognormal curve (blue line). (C) Representative trajectories of AMPAR molecules on somas and neurites. Scale bar = 2 μm. (D) The mean square displacement functions and trajectories represent AMPAR molecules with Brownian motion (red) and confined motion (blue). Scale bar = 0.1 μm. (E,F) The cumulative probability functions of D values of AMPAR (E) and mGluR1 (F) on neurites and somas ( n = 510–676 trajectories). *** p < 0.001.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

    doi: 10.3389/fcell.2021.708715

    Figure Lengend Snippet: Characterization of differentiated PC12 cells and validation of single-molecule labeling. (A,B) Left, Intensity profiles of a single ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) signal. The arrows indicate single-step photobleaching. Right, Histogram showing the intensity value of every spot found in a recording of ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) , superimposed with a single fitted lognormal curve (blue line). (C) Representative trajectories of AMPAR molecules on somas and neurites. Scale bar = 2 μm. (D) The mean square displacement functions and trajectories represent AMPAR molecules with Brownian motion (red) and confined motion (blue). Scale bar = 0.1 μm. (E,F) The cumulative probability functions of D values of AMPAR (E) and mGluR1 (F) on neurites and somas ( n = 510–676 trajectories). *** p < 0.001.

    Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

    Techniques: Labeling

    Effect of E2 on the surface movement of GluR2-AMPAR and mGluR1. (A) Effect of different concentrations of E2 on the diffusion coefficient (D, μm 2 /s) of GluR2-AMPAR (A) and mGluR1 (B) (% of vehicle treatment as the mean ± SEM, n = 425–1145 trajectories per group). (C,D) Line graphs depict changes in D of GluR2-AMPAR (C) and mGluR1 (D) molecules at different time points after the administration of the most effective concentration of E2 (% of vehicle treatment as the mean D ± SEM, n = 117–187 trajectories per time point). * p < 0.05; ** p < 0.01; *** p < 0.001.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

    doi: 10.3389/fcell.2021.708715

    Figure Lengend Snippet: Effect of E2 on the surface movement of GluR2-AMPAR and mGluR1. (A) Effect of different concentrations of E2 on the diffusion coefficient (D, μm 2 /s) of GluR2-AMPAR (A) and mGluR1 (B) (% of vehicle treatment as the mean ± SEM, n = 425–1145 trajectories per group). (C,D) Line graphs depict changes in D of GluR2-AMPAR (C) and mGluR1 (D) molecules at different time points after the administration of the most effective concentration of E2 (% of vehicle treatment as the mean D ± SEM, n = 117–187 trajectories per time point). * p < 0.05; ** p < 0.01; *** p < 0.001.

    Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

    Techniques: Diffusion-based Assay, Concentration Assay

    Effect of estrogen receptor modulation on the surface movement of GluR2-AMPAR. (A) Representative PCR gel electrophoresis image depicting the expression of estrogen receptor beta (ERβ) and G protein-coupled estrogen receptor 1 (GPER1) mRNA in dPC12. Estrogen receptor alpha (ERα) mRNA was not detected. (B) Histograms demonstrate the mean D AMPAR as a percentage of vehicle control on somas and neurites in the presence of the estrogen receptor, β (ERβ) agonist diarylpropionitrile (DPN), a GPER1 agonist (G1), G1+DPN together, a GPER1 antagonist (G15) and G15+E2 (with 100 pM of E2 on the somas and 100 nM of E2 on the neurites) (mean ± SEM; n = 215–641 trajectories). *** p < 0.001.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

    doi: 10.3389/fcell.2021.708715

    Figure Lengend Snippet: Effect of estrogen receptor modulation on the surface movement of GluR2-AMPAR. (A) Representative PCR gel electrophoresis image depicting the expression of estrogen receptor beta (ERβ) and G protein-coupled estrogen receptor 1 (GPER1) mRNA in dPC12. Estrogen receptor alpha (ERα) mRNA was not detected. (B) Histograms demonstrate the mean D AMPAR as a percentage of vehicle control on somas and neurites in the presence of the estrogen receptor, β (ERβ) agonist diarylpropionitrile (DPN), a GPER1 agonist (G1), G1+DPN together, a GPER1 antagonist (G15) and G15+E2 (with 100 pM of E2 on the somas and 100 nM of E2 on the neurites) (mean ± SEM; n = 215–641 trajectories). *** p < 0.001.

    Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

    Techniques: Nucleic Acid Electrophoresis, Expressing

    The GluR2-AMPAR/GPER1 ratio and molecular distance between GPER1 and GluR2-AMPAR in the membrane. (A) STORM images depicting immunolabeled AMPAR (magenta) and GPER1 (cyan) molecules on dPC12. Dashed lines delineate the borders of the neurites and somas. Scale bar = 2 μm; inset Scale bar = 0.5 μm. (B) The ratio between the number of GPER1 and AMPAR molecules (GPER1/GluR2-AMPAR) on the neurites and somas ( n = 11 somas or neurites). (C1) Photomicrographs depict GPER1 immunoreactivity (visualized with STED microscopy) in dPC12 after 10 min of vehicle (left) or of 100 nM of E2 treatment (right). Scale bar = 2 μm. (C2) One 2 μm 2 (between parallel white bars) and one 10 μm 2 (to the left) areas were selected within each ROI for the membrane and cytoplasmic regions of each cell, respectively. Integrated density was calculated and normalized to the area. Scale bar = 0.5 μm. (D) Dual labeling of plasma membrane and GPER1 molecules defines the membrane regions (approximately 1 μm wide). Scale bar = 0.5 μm. (E) Line graph of the fluorescent intensity calculated from the magnified STED inserts (C2). (F) Integrated density graphs of GPER1 show the effect of vehicle and 100 nM of E2 treatment in the membrane and in the cytoplasm ( n = 15 cells were evaluated in each group). * p < 0.05.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

    doi: 10.3389/fcell.2021.708715

    Figure Lengend Snippet: The GluR2-AMPAR/GPER1 ratio and molecular distance between GPER1 and GluR2-AMPAR in the membrane. (A) STORM images depicting immunolabeled AMPAR (magenta) and GPER1 (cyan) molecules on dPC12. Dashed lines delineate the borders of the neurites and somas. Scale bar = 2 μm; inset Scale bar = 0.5 μm. (B) The ratio between the number of GPER1 and AMPAR molecules (GPER1/GluR2-AMPAR) on the neurites and somas ( n = 11 somas or neurites). (C1) Photomicrographs depict GPER1 immunoreactivity (visualized with STED microscopy) in dPC12 after 10 min of vehicle (left) or of 100 nM of E2 treatment (right). Scale bar = 2 μm. (C2) One 2 μm 2 (between parallel white bars) and one 10 μm 2 (to the left) areas were selected within each ROI for the membrane and cytoplasmic regions of each cell, respectively. Integrated density was calculated and normalized to the area. Scale bar = 0.5 μm. (D) Dual labeling of plasma membrane and GPER1 molecules defines the membrane regions (approximately 1 μm wide). Scale bar = 0.5 μm. (E) Line graph of the fluorescent intensity calculated from the magnified STED inserts (C2). (F) Integrated density graphs of GPER1 show the effect of vehicle and 100 nM of E2 treatment in the membrane and in the cytoplasm ( n = 15 cells were evaluated in each group). * p < 0.05.

    Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

    Techniques: Immunolabeling, Microscopy, Labeling

    The role of the cortical actin in the rapid effect of E2. (A) Left, confocal images depict Alexa Fluor 488 phalloidin-labeled cortical actin network in dPC12 after treatment with vehicle, 1 μM of latA, 1 μM of SP600125 or 1 μM of GSK429286. Scale bar = 5 μm; insert Scale bar = 0.5 μm. Right, the bar graph shows the effect of LatA, GSK429286, and SP600125 on the integrated density of the fluorescently labeled cortical actin network [ n = 3 cells per group (3 ROIs per cell)]. (B1,B2) Effect of LatA, GSK429286, and SP600125 treatment on D AMPAR (% of vehicle treatment as the mean ± SEM; n = 215–544 trajectories). (C1,C2) Effect of 100 pM of E2 on somas and 100 nM of E2 on neurites with or without LatA, GSK429286, and SP600125 (% of vehicle treatment as the mean ± SEM; n = 184–277 trajectories). *** p < 0.001.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

    doi: 10.3389/fcell.2021.708715

    Figure Lengend Snippet: The role of the cortical actin in the rapid effect of E2. (A) Left, confocal images depict Alexa Fluor 488 phalloidin-labeled cortical actin network in dPC12 after treatment with vehicle, 1 μM of latA, 1 μM of SP600125 or 1 μM of GSK429286. Scale bar = 5 μm; insert Scale bar = 0.5 μm. Right, the bar graph shows the effect of LatA, GSK429286, and SP600125 on the integrated density of the fluorescently labeled cortical actin network [ n = 3 cells per group (3 ROIs per cell)]. (B1,B2) Effect of LatA, GSK429286, and SP600125 treatment on D AMPAR (% of vehicle treatment as the mean ± SEM; n = 215–544 trajectories). (C1,C2) Effect of 100 pM of E2 on somas and 100 nM of E2 on neurites with or without LatA, GSK429286, and SP600125 (% of vehicle treatment as the mean ± SEM; n = 184–277 trajectories). *** p < 0.001.

    Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

    Techniques: Labeling

    Effect of E2 on the surface movement of GluR2-AMPA on primary hippocampal neurons. (A) Photomicrograph shows a primary hippocampal neuron labeled with homer-1 (synapse) and β-III tubulin (neuron). Scale bar = 10 μm, insert Scale bar = 2 μm. (B) Dual color STED image of a hippocampal neuron overlayed to differential interference contrast microscopy image depicts live-cell synapse labeling MitoTracker Deep Red (red) and presynaptic protein bassoon (green). Scale bar = 1 μm. (C) Distribution of D values of extrasynaptic and synaptic GluR2-AMPAR under control conditions (median ± IQR, n = 754 extrasynaptic trajectories and n = 104 synaptic trajectories). (D) Effect of E2 (100 pM and 100 nM) on D of extrasynaptic and synaptic GluR2-AMPA with or without chemical LTP (cLTP) induced by glycine/picrotoxin (gly/pic) (% of vehicle treatment as the mean ± SEM; n = 742–928 extrasynaptic trajectories and n = 104–155 synaptic trajectories). (E,F) Effect of vehicle, E2 (100 n, 100 pM) with or without cLTP (gly/pic) on synaptic dwell time (mean ± SEM (s); n = 104–155) (E) and relative surface distribution of synaptic GluR2-AMPAR content (synaptic/total GluR2-AMPA molecule trajectories) (mean ± SEM, n = 8–18 recordings) (F) . * p < 0.05; ** p < 0.01; *** p < 0.001.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

    doi: 10.3389/fcell.2021.708715

    Figure Lengend Snippet: Effect of E2 on the surface movement of GluR2-AMPA on primary hippocampal neurons. (A) Photomicrograph shows a primary hippocampal neuron labeled with homer-1 (synapse) and β-III tubulin (neuron). Scale bar = 10 μm, insert Scale bar = 2 μm. (B) Dual color STED image of a hippocampal neuron overlayed to differential interference contrast microscopy image depicts live-cell synapse labeling MitoTracker Deep Red (red) and presynaptic protein bassoon (green). Scale bar = 1 μm. (C) Distribution of D values of extrasynaptic and synaptic GluR2-AMPAR under control conditions (median ± IQR, n = 754 extrasynaptic trajectories and n = 104 synaptic trajectories). (D) Effect of E2 (100 pM and 100 nM) on D of extrasynaptic and synaptic GluR2-AMPA with or without chemical LTP (cLTP) induced by glycine/picrotoxin (gly/pic) (% of vehicle treatment as the mean ± SEM; n = 742–928 extrasynaptic trajectories and n = 104–155 synaptic trajectories). (E,F) Effect of vehicle, E2 (100 n, 100 pM) with or without cLTP (gly/pic) on synaptic dwell time (mean ± SEM (s); n = 104–155) (E) and relative surface distribution of synaptic GluR2-AMPAR content (synaptic/total GluR2-AMPA molecule trajectories) (mean ± SEM, n = 8–18 recordings) (F) . * p < 0.05; ** p < 0.01; *** p < 0.001.

    Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

    Techniques: Labeling, Microscopy