anti pan mglu5  (Alomone Labs)


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    Structured Review

    Alomone Labs anti pan mglu5
    Group 1 mGluRs interact with Actn4. A , schematic of wild type Actn4 and deletion mutants fused to GST used in a pulldown assay with mGlu 1b . B , the Actn4 CH region is necessary and sufficient for mGlu 1b binding. Shown are representative immunoblots ( IB )of a pulldown assay between GST-Actn4 fusion proteins from A and lysates of HEK293 cells transfected with mGlu 1b , probed with anti-mGlu 1b , and stained with Ponceau. C and D , the mGlu 1 cytoplasmic tail is sufficient for Actn4 binding. Shown is a schematic of chimeric constructs of VSVG protein fused to mGlu 1b or mGlu 1a carboxyl tails. Yellow highlights the membrane-proximal region of the tail that is conserved between receptor isoforms (CR region, see below). Also shown are representative immunoblots of input (20 μg) and pulldown (input lysate, 2 mg) between GST-Actn4 and VSVG-mGlu 1a-Tail ( D ) and VSVG-mGlu 1b-Tail ( C ) probed with anti-mGlu 1a or anti-mGlu 1b antibody, respectively. The estimated pulldown fraction in vitro is ∼1% of input receptors. E , Actn4 interacts directly with the CR region of the mGlu 1 intracellular tail. Shown is a schematic of the CR construct and immunoblots illustrating in vitro binding of a purified His-tagged CR fragment and GST-tagged full-length Actn4 ( GST-Actn4 , left panel ) or Actn4 CH region ( GST-Actn4 1–296 , right panel ). Shown are bound and unbound (14% of input GST-tagged proteins) fractions probed with anti-GST and anti-His tag antibodies. The arrow points to full-length GST-Actn4 ( left panel ). Lower molecular mass bands were derived from partial protein fragmentation. The estimated relative bound fractions are 40% and 26% of input for GST-Actn4 and GST-Actn41–296, respectively. F , Actn4 coprecipitates with mGlu 5 in the brain. Shown are representative immunoblots of input lysate and precipitated proteins (from 2.5 mg of input lysate) probed with anti-Actn4 (input, 10 μg) and anti-mGlu 5 (input, 40 μg). The estimated immunoprecipitation (IP) is 5% and 3% of input for <t>mGlu5</t> and Actn4, respectively.
    Anti Pan Mglu5, 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/anti pan mglu5/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti pan mglu5 - by Bioz Stars, 2021-12
    94/100 stars

    Images

    1) Product Images from "Actinin-4 Governs Dendritic Spine Dynamics and Promotes Their Remodeling by Metabotropic Glutamate Receptors *"

    Article Title: Actinin-4 Governs Dendritic Spine Dynamics and Promotes Their Remodeling by Metabotropic Glutamate Receptors *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.640136

    Group 1 mGluRs interact with Actn4. A , schematic of wild type Actn4 and deletion mutants fused to GST used in a pulldown assay with mGlu 1b . B , the Actn4 CH region is necessary and sufficient for mGlu 1b binding. Shown are representative immunoblots ( IB )of a pulldown assay between GST-Actn4 fusion proteins from A and lysates of HEK293 cells transfected with mGlu 1b , probed with anti-mGlu 1b , and stained with Ponceau. C and D , the mGlu 1 cytoplasmic tail is sufficient for Actn4 binding. Shown is a schematic of chimeric constructs of VSVG protein fused to mGlu 1b or mGlu 1a carboxyl tails. Yellow highlights the membrane-proximal region of the tail that is conserved between receptor isoforms (CR region, see below). Also shown are representative immunoblots of input (20 μg) and pulldown (input lysate, 2 mg) between GST-Actn4 and VSVG-mGlu 1a-Tail ( D ) and VSVG-mGlu 1b-Tail ( C ) probed with anti-mGlu 1a or anti-mGlu 1b antibody, respectively. The estimated pulldown fraction in vitro is ∼1% of input receptors. E , Actn4 interacts directly with the CR region of the mGlu 1 intracellular tail. Shown is a schematic of the CR construct and immunoblots illustrating in vitro binding of a purified His-tagged CR fragment and GST-tagged full-length Actn4 ( GST-Actn4 , left panel ) or Actn4 CH region ( GST-Actn4 1–296 , right panel ). Shown are bound and unbound (14% of input GST-tagged proteins) fractions probed with anti-GST and anti-His tag antibodies. The arrow points to full-length GST-Actn4 ( left panel ). Lower molecular mass bands were derived from partial protein fragmentation. The estimated relative bound fractions are 40% and 26% of input for GST-Actn4 and GST-Actn41–296, respectively. F , Actn4 coprecipitates with mGlu 5 in the brain. Shown are representative immunoblots of input lysate and precipitated proteins (from 2.5 mg of input lysate) probed with anti-Actn4 (input, 10 μg) and anti-mGlu 5 (input, 40 μg). The estimated immunoprecipitation (IP) is 5% and 3% of input for mGlu5 and Actn4, respectively.
    Figure Legend Snippet: Group 1 mGluRs interact with Actn4. A , schematic of wild type Actn4 and deletion mutants fused to GST used in a pulldown assay with mGlu 1b . B , the Actn4 CH region is necessary and sufficient for mGlu 1b binding. Shown are representative immunoblots ( IB )of a pulldown assay between GST-Actn4 fusion proteins from A and lysates of HEK293 cells transfected with mGlu 1b , probed with anti-mGlu 1b , and stained with Ponceau. C and D , the mGlu 1 cytoplasmic tail is sufficient for Actn4 binding. Shown is a schematic of chimeric constructs of VSVG protein fused to mGlu 1b or mGlu 1a carboxyl tails. Yellow highlights the membrane-proximal region of the tail that is conserved between receptor isoforms (CR region, see below). Also shown are representative immunoblots of input (20 μg) and pulldown (input lysate, 2 mg) between GST-Actn4 and VSVG-mGlu 1a-Tail ( D ) and VSVG-mGlu 1b-Tail ( C ) probed with anti-mGlu 1a or anti-mGlu 1b antibody, respectively. The estimated pulldown fraction in vitro is ∼1% of input receptors. E , Actn4 interacts directly with the CR region of the mGlu 1 intracellular tail. Shown is a schematic of the CR construct and immunoblots illustrating in vitro binding of a purified His-tagged CR fragment and GST-tagged full-length Actn4 ( GST-Actn4 , left panel ) or Actn4 CH region ( GST-Actn4 1–296 , right panel ). Shown are bound and unbound (14% of input GST-tagged proteins) fractions probed with anti-GST and anti-His tag antibodies. The arrow points to full-length GST-Actn4 ( left panel ). Lower molecular mass bands were derived from partial protein fragmentation. The estimated relative bound fractions are 40% and 26% of input for GST-Actn4 and GST-Actn41–296, respectively. F , Actn4 coprecipitates with mGlu 5 in the brain. Shown are representative immunoblots of input lysate and precipitated proteins (from 2.5 mg of input lysate) probed with anti-Actn4 (input, 10 μg) and anti-mGlu 5 (input, 40 μg). The estimated immunoprecipitation (IP) is 5% and 3% of input for mGlu5 and Actn4, respectively.

    Techniques Used: Binding Assay, Western Blot, Transfection, Staining, Construct, In Vitro, Purification, Derivative Assay, Immunoprecipitation

    2) Product Images from "Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse"

    Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039585

    VE-2B interacted directly with SAP102, and VE-2B, SAP102 and PSD-95 formed co-clusters. (A) Western blotting was performed on cortical neurons infected with VE-2B and immunoprecipitated with i1 antibody to VSVG at 10 minutes and 3 hours after switch to permissive temperature (24 fold enrichment). Immunoblots were performed with rabbit anti-VSVG, PSD-95, and SAP102. Input, unbound, and immunoprecipitation fractions (IP) were run for each blot. To minimize the possibility of false-positives, buffer only was added to the lanes adjacent to the VE-2B IP lanes. Identical film exposure times for both anti-PSD-95 and anti-SAP102 indicate that SAP102 associates at 10 minutes and 3 hours after ER exit whereas PSD-95 does not. Longer film exposure times did not unambiguously indicate that PSD-95 was directly associated. (B) Western blotting was performed on cortical neurons infected with VE and immunoprecipitated with antibody to VSVG at 10 minutes after switch to permissive temperature. Immunoblots were then performed with rabbit anti-VSVG, and SAP102. Input, unbound, and IP fractions were run for each blot. The results indicated that VE (left panel) did not co-immunoprecipitate measurable quantities of SAP102 (right panel). (C) VE-2B transfected neurons 3 hours after ER release were immunostained for PSD-95 (red) and SAP102 (blue; scale bar, 5 µm). Aqua-colored arrows indicate VE-2B co-localized with SAP102 primarily. White arrows indicate VE-2B co-localized with both PSD-95 and SAP102.
    Figure Legend Snippet: VE-2B interacted directly with SAP102, and VE-2B, SAP102 and PSD-95 formed co-clusters. (A) Western blotting was performed on cortical neurons infected with VE-2B and immunoprecipitated with i1 antibody to VSVG at 10 minutes and 3 hours after switch to permissive temperature (24 fold enrichment). Immunoblots were performed with rabbit anti-VSVG, PSD-95, and SAP102. Input, unbound, and immunoprecipitation fractions (IP) were run for each blot. To minimize the possibility of false-positives, buffer only was added to the lanes adjacent to the VE-2B IP lanes. Identical film exposure times for both anti-PSD-95 and anti-SAP102 indicate that SAP102 associates at 10 minutes and 3 hours after ER exit whereas PSD-95 does not. Longer film exposure times did not unambiguously indicate that PSD-95 was directly associated. (B) Western blotting was performed on cortical neurons infected with VE and immunoprecipitated with antibody to VSVG at 10 minutes after switch to permissive temperature. Immunoblots were then performed with rabbit anti-VSVG, and SAP102. Input, unbound, and IP fractions were run for each blot. The results indicated that VE (left panel) did not co-immunoprecipitate measurable quantities of SAP102 (right panel). (C) VE-2B transfected neurons 3 hours after ER release were immunostained for PSD-95 (red) and SAP102 (blue; scale bar, 5 µm). Aqua-colored arrows indicate VE-2B co-localized with SAP102 primarily. White arrows indicate VE-2B co-localized with both PSD-95 and SAP102.

    Techniques Used: Western Blot, Infection, Immunoprecipitation, Transfection

    VE-2B co-localized with PSD-95 from the TGN to the plasma membrane. (A) At 10 minutes after release from the ER, VE-2B showed little colocalization with antibody staining to endogenous PSD-95 in the soma (top panels; scale bars are 10 µm). Forty-five minutes after ER release (second row), a time point at which the leading edge of VE-2B reaches the surface, PSD-95 showed significant colocalization with VE-2B. Similar results were obtained with VE-2A (see Table S1 ). (B) Colocalization with PSD-95 was also evident in dendrites at 45 minutes and later time points, such as 3 hours (see Fig. 6). VE-2B (top panel) showed co-localization with endogenous PSD-95 (middle panel) in dendrites at 45 minutes after permissive temperature, and later (scale bar 5 µm). (C) Neurons transfected with VE-2B were subjected to 40°C then the medium was exchanged with medium equilibrated at 20°C. Cultures were maintained at 20°C for 1 hour to allow VE-2B cargo exiting the ER to build up in the TGN. Immunostaining for endogenous TGN38 (a TGN marker; top panels) and PSD-95 (second row) in VE-2B transfected neurons subjected to the 20°C temperature manipulation showed robust colocalization (scale bars 5 µm). (D) Quantification of PSD-95 co-localization at different stages of the secretory pathway. There was a significant difference among the groups using a one-way Anova. However, post hoc comparisons demonstrated an insignificant pairwise difference at 10 minutes following ER exit. PSD-95 showed levels of co-localization with VE-2B (open circles) not significantly different from those of VE-2BΔ7 (filled triangles) 10 minutes after ER release. Forty-five minutes after ER exit, VE-2B co-localization with PSD-95 (filled circles) was significantly enhanced at most thresholds when compared to VE-2B colocalization at 10 minutes after ER exit. The significant increase in colocalization of VE-2B with PSD-95 seen at 45 minutes after ER exit could be reproduced by switching media to 20°C for 1 hour (open squares). To verify that VE-2B was concentrated in the TGN after 1 hour 20°C incubation, colocalization of VE-2B with TGN38 was quantified (filled squares).
    Figure Legend Snippet: VE-2B co-localized with PSD-95 from the TGN to the plasma membrane. (A) At 10 minutes after release from the ER, VE-2B showed little colocalization with antibody staining to endogenous PSD-95 in the soma (top panels; scale bars are 10 µm). Forty-five minutes after ER release (second row), a time point at which the leading edge of VE-2B reaches the surface, PSD-95 showed significant colocalization with VE-2B. Similar results were obtained with VE-2A (see Table S1 ). (B) Colocalization with PSD-95 was also evident in dendrites at 45 minutes and later time points, such as 3 hours (see Fig. 6). VE-2B (top panel) showed co-localization with endogenous PSD-95 (middle panel) in dendrites at 45 minutes after permissive temperature, and later (scale bar 5 µm). (C) Neurons transfected with VE-2B were subjected to 40°C then the medium was exchanged with medium equilibrated at 20°C. Cultures were maintained at 20°C for 1 hour to allow VE-2B cargo exiting the ER to build up in the TGN. Immunostaining for endogenous TGN38 (a TGN marker; top panels) and PSD-95 (second row) in VE-2B transfected neurons subjected to the 20°C temperature manipulation showed robust colocalization (scale bars 5 µm). (D) Quantification of PSD-95 co-localization at different stages of the secretory pathway. There was a significant difference among the groups using a one-way Anova. However, post hoc comparisons demonstrated an insignificant pairwise difference at 10 minutes following ER exit. PSD-95 showed levels of co-localization with VE-2B (open circles) not significantly different from those of VE-2BΔ7 (filled triangles) 10 minutes after ER release. Forty-five minutes after ER exit, VE-2B co-localization with PSD-95 (filled circles) was significantly enhanced at most thresholds when compared to VE-2B colocalization at 10 minutes after ER exit. The significant increase in colocalization of VE-2B with PSD-95 seen at 45 minutes after ER exit could be reproduced by switching media to 20°C for 1 hour (open squares). To verify that VE-2B was concentrated in the TGN after 1 hour 20°C incubation, colocalization of VE-2B with TGN38 was quantified (filled squares).

    Techniques Used: Staining, Transfection, Immunostaining, Marker, Incubation

    VE-2B and the MAGUKs are consistent with co-transportation to the synapse. (A) VE-2B chimeras co-localized with high intensity PSD-95 (left graph) and SAP102 (right graph) in dendrites 3 hours after release with the red threshold fixed at 4X background (104–255 inclusive gray scale). Colocalization was quantified as described in Experimental Methods. VE, VE-2B, and VE-2BΔ7 colocalization was assessed across the range of inclusive thresholds for green indicated on the x-axis. There was a significant group effect by one-way Anova even at even the lowest green threshold (p
    Figure Legend Snippet: VE-2B and the MAGUKs are consistent with co-transportation to the synapse. (A) VE-2B chimeras co-localized with high intensity PSD-95 (left graph) and SAP102 (right graph) in dendrites 3 hours after release with the red threshold fixed at 4X background (104–255 inclusive gray scale). Colocalization was quantified as described in Experimental Methods. VE, VE-2B, and VE-2BΔ7 colocalization was assessed across the range of inclusive thresholds for green indicated on the x-axis. There was a significant group effect by one-way Anova even at even the lowest green threshold (p

    Techniques Used:

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    Alomone Labs anti pan mglu5
    Group 1 mGluRs interact with Actn4. A , schematic of wild type Actn4 and deletion mutants fused to GST used in a pulldown assay with mGlu 1b . B , the Actn4 CH region is necessary and sufficient for mGlu 1b binding. Shown are representative immunoblots ( IB )of a pulldown assay between GST-Actn4 fusion proteins from A and lysates of HEK293 cells transfected with mGlu 1b , probed with anti-mGlu 1b , and stained with Ponceau. C and D , the mGlu 1 cytoplasmic tail is sufficient for Actn4 binding. Shown is a schematic of chimeric constructs of VSVG protein fused to mGlu 1b or mGlu 1a carboxyl tails. Yellow highlights the membrane-proximal region of the tail that is conserved between receptor isoforms (CR region, see below). Also shown are representative immunoblots of input (20 μg) and pulldown (input lysate, 2 mg) between GST-Actn4 and VSVG-mGlu 1a-Tail ( D ) and VSVG-mGlu 1b-Tail ( C ) probed with anti-mGlu 1a or anti-mGlu 1b antibody, respectively. The estimated pulldown fraction in vitro is ∼1% of input receptors. E , Actn4 interacts directly with the CR region of the mGlu 1 intracellular tail. Shown is a schematic of the CR construct and immunoblots illustrating in vitro binding of a purified His-tagged CR fragment and GST-tagged full-length Actn4 ( GST-Actn4 , left panel ) or Actn4 CH region ( GST-Actn4 1–296 , right panel ). Shown are bound and unbound (14% of input GST-tagged proteins) fractions probed with anti-GST and anti-His tag antibodies. The arrow points to full-length GST-Actn4 ( left panel ). Lower molecular mass bands were derived from partial protein fragmentation. The estimated relative bound fractions are 40% and 26% of input for GST-Actn4 and GST-Actn41–296, respectively. F , Actn4 coprecipitates with mGlu 5 in the brain. Shown are representative immunoblots of input lysate and precipitated proteins (from 2.5 mg of input lysate) probed with anti-Actn4 (input, 10 μg) and anti-mGlu 5 (input, 40 μg). The estimated immunoprecipitation (IP) is 5% and 3% of input for <t>mGlu5</t> and Actn4, respectively.
    Anti Pan Mglu5, 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/anti pan mglu5/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti pan mglu5 - by Bioz Stars, 2021-12
    94/100 stars
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    86
    Alomone Labs anti psd 95 antibody
    <t>PSD-95</t> expression pattern depends on culture duration. ( A , B ) Representative tdTomato-positive hRGCs ( red ) cultured for 1 ( A ) and 4 ( B ) weeks and immunolabeled against PSD-95 ( green ). Scale bar = 20 µm. ( C ) PSD-95 labeling within hRGC somas significantly decreased after 4 weeks versus earlier time points ( P ≤ 0.033). ( D ) Quantification of PSD-95 labeling within hRGC primary neurites. PSD-95 labeling significantly increased by 3 and 4 weeks in culture compared with 1-week cells. Significance indicators: * comparison with 1-week cells, # comparison with 2-week cells, ^ comparison with 3-week cells. Statistics: one-way analysis of variance, Tukey post hoc test ( C , D ). Data are mean ± standard error of the mean.
    Anti Psd 95 Antibody, 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
    https://www.bioz.com/result/anti psd 95 antibody/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti psd 95 antibody - by Bioz Stars, 2021-12
    86/100 stars
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    Group 1 mGluRs interact with Actn4. A , schematic of wild type Actn4 and deletion mutants fused to GST used in a pulldown assay with mGlu 1b . B , the Actn4 CH region is necessary and sufficient for mGlu 1b binding. Shown are representative immunoblots ( IB )of a pulldown assay between GST-Actn4 fusion proteins from A and lysates of HEK293 cells transfected with mGlu 1b , probed with anti-mGlu 1b , and stained with Ponceau. C and D , the mGlu 1 cytoplasmic tail is sufficient for Actn4 binding. Shown is a schematic of chimeric constructs of VSVG protein fused to mGlu 1b or mGlu 1a carboxyl tails. Yellow highlights the membrane-proximal region of the tail that is conserved between receptor isoforms (CR region, see below). Also shown are representative immunoblots of input (20 μg) and pulldown (input lysate, 2 mg) between GST-Actn4 and VSVG-mGlu 1a-Tail ( D ) and VSVG-mGlu 1b-Tail ( C ) probed with anti-mGlu 1a or anti-mGlu 1b antibody, respectively. The estimated pulldown fraction in vitro is ∼1% of input receptors. E , Actn4 interacts directly with the CR region of the mGlu 1 intracellular tail. Shown is a schematic of the CR construct and immunoblots illustrating in vitro binding of a purified His-tagged CR fragment and GST-tagged full-length Actn4 ( GST-Actn4 , left panel ) or Actn4 CH region ( GST-Actn4 1–296 , right panel ). Shown are bound and unbound (14% of input GST-tagged proteins) fractions probed with anti-GST and anti-His tag antibodies. The arrow points to full-length GST-Actn4 ( left panel ). Lower molecular mass bands were derived from partial protein fragmentation. The estimated relative bound fractions are 40% and 26% of input for GST-Actn4 and GST-Actn41–296, respectively. F , Actn4 coprecipitates with mGlu 5 in the brain. Shown are representative immunoblots of input lysate and precipitated proteins (from 2.5 mg of input lysate) probed with anti-Actn4 (input, 10 μg) and anti-mGlu 5 (input, 40 μg). The estimated immunoprecipitation (IP) is 5% and 3% of input for mGlu5 and Actn4, respectively.

    Journal: The Journal of Biological Chemistry

    Article Title: Actinin-4 Governs Dendritic Spine Dynamics and Promotes Their Remodeling by Metabotropic Glutamate Receptors *

    doi: 10.1074/jbc.M115.640136

    Figure Lengend Snippet: Group 1 mGluRs interact with Actn4. A , schematic of wild type Actn4 and deletion mutants fused to GST used in a pulldown assay with mGlu 1b . B , the Actn4 CH region is necessary and sufficient for mGlu 1b binding. Shown are representative immunoblots ( IB )of a pulldown assay between GST-Actn4 fusion proteins from A and lysates of HEK293 cells transfected with mGlu 1b , probed with anti-mGlu 1b , and stained with Ponceau. C and D , the mGlu 1 cytoplasmic tail is sufficient for Actn4 binding. Shown is a schematic of chimeric constructs of VSVG protein fused to mGlu 1b or mGlu 1a carboxyl tails. Yellow highlights the membrane-proximal region of the tail that is conserved between receptor isoforms (CR region, see below). Also shown are representative immunoblots of input (20 μg) and pulldown (input lysate, 2 mg) between GST-Actn4 and VSVG-mGlu 1a-Tail ( D ) and VSVG-mGlu 1b-Tail ( C ) probed with anti-mGlu 1a or anti-mGlu 1b antibody, respectively. The estimated pulldown fraction in vitro is ∼1% of input receptors. E , Actn4 interacts directly with the CR region of the mGlu 1 intracellular tail. Shown is a schematic of the CR construct and immunoblots illustrating in vitro binding of a purified His-tagged CR fragment and GST-tagged full-length Actn4 ( GST-Actn4 , left panel ) or Actn4 CH region ( GST-Actn4 1–296 , right panel ). Shown are bound and unbound (14% of input GST-tagged proteins) fractions probed with anti-GST and anti-His tag antibodies. The arrow points to full-length GST-Actn4 ( left panel ). Lower molecular mass bands were derived from partial protein fragmentation. The estimated relative bound fractions are 40% and 26% of input for GST-Actn4 and GST-Actn41–296, respectively. F , Actn4 coprecipitates with mGlu 5 in the brain. Shown are representative immunoblots of input lysate and precipitated proteins (from 2.5 mg of input lysate) probed with anti-Actn4 (input, 10 μg) and anti-mGlu 5 (input, 40 μg). The estimated immunoprecipitation (IP) is 5% and 3% of input for mGlu5 and Actn4, respectively.

    Article Snippet: The following antibodies were used: goat polyclonal anti-GAPDH (GenScript); chicken polyclonal anti-MAP2 (EnCor Biotech); the rabbit monoclonal antibodies anti-Actn4, anti-Actn1, and anti-mGlu5 (Epitomics); the rabbit polyclonal antibodies anti-GFP (Santa Cruz Biotechnology), anti-pan-mGlu1 , anti-pan-mGlu5 (Alomone Labs), anti-mGlu5 (GenScript), anti-PSD95 (Zymed Laboratories Inc.), anti-Actn4 (Enzo Life Sciences), anti-phospho-ERK1/2(Thr-202/Tyr-204), anti-ERK1/2, anti-phospho-CaMKII(Thr-286/Thr-287), and anti-CaMKII (Cell Signaling Technology); and the mouse monoclonal antibodies anti-MAP2 clone AP20 (Roche Applied Science), anti-actinin clone EA-53 (Sigma-Aldrich), anti-Actn4 (Abnova), anti-mGlu1a (BD Biosciences), anti-PSD95 clone K28/43 (University of California Davis/National Institutes of Health NeuroMab Facility), and anti-His tag (Aviva Systems Biology).

    Techniques: Binding Assay, Western Blot, Transfection, Staining, Construct, In Vitro, Purification, Derivative Assay, Immunoprecipitation

    VE-2B interacted directly with SAP102, and VE-2B, SAP102 and PSD-95 formed co-clusters. (A) Western blotting was performed on cortical neurons infected with VE-2B and immunoprecipitated with i1 antibody to VSVG at 10 minutes and 3 hours after switch to permissive temperature (24 fold enrichment). Immunoblots were performed with rabbit anti-VSVG, PSD-95, and SAP102. Input, unbound, and immunoprecipitation fractions (IP) were run for each blot. To minimize the possibility of false-positives, buffer only was added to the lanes adjacent to the VE-2B IP lanes. Identical film exposure times for both anti-PSD-95 and anti-SAP102 indicate that SAP102 associates at 10 minutes and 3 hours after ER exit whereas PSD-95 does not. Longer film exposure times did not unambiguously indicate that PSD-95 was directly associated. (B) Western blotting was performed on cortical neurons infected with VE and immunoprecipitated with antibody to VSVG at 10 minutes after switch to permissive temperature. Immunoblots were then performed with rabbit anti-VSVG, and SAP102. Input, unbound, and IP fractions were run for each blot. The results indicated that VE (left panel) did not co-immunoprecipitate measurable quantities of SAP102 (right panel). (C) VE-2B transfected neurons 3 hours after ER release were immunostained for PSD-95 (red) and SAP102 (blue; scale bar, 5 µm). Aqua-colored arrows indicate VE-2B co-localized with SAP102 primarily. White arrows indicate VE-2B co-localized with both PSD-95 and SAP102.

    Journal: PLoS ONE

    Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

    doi: 10.1371/journal.pone.0039585

    Figure Lengend Snippet: VE-2B interacted directly with SAP102, and VE-2B, SAP102 and PSD-95 formed co-clusters. (A) Western blotting was performed on cortical neurons infected with VE-2B and immunoprecipitated with i1 antibody to VSVG at 10 minutes and 3 hours after switch to permissive temperature (24 fold enrichment). Immunoblots were performed with rabbit anti-VSVG, PSD-95, and SAP102. Input, unbound, and immunoprecipitation fractions (IP) were run for each blot. To minimize the possibility of false-positives, buffer only was added to the lanes adjacent to the VE-2B IP lanes. Identical film exposure times for both anti-PSD-95 and anti-SAP102 indicate that SAP102 associates at 10 minutes and 3 hours after ER exit whereas PSD-95 does not. Longer film exposure times did not unambiguously indicate that PSD-95 was directly associated. (B) Western blotting was performed on cortical neurons infected with VE and immunoprecipitated with antibody to VSVG at 10 minutes after switch to permissive temperature. Immunoblots were then performed with rabbit anti-VSVG, and SAP102. Input, unbound, and IP fractions were run for each blot. The results indicated that VE (left panel) did not co-immunoprecipitate measurable quantities of SAP102 (right panel). (C) VE-2B transfected neurons 3 hours after ER release were immunostained for PSD-95 (red) and SAP102 (blue; scale bar, 5 µm). Aqua-colored arrows indicate VE-2B co-localized with SAP102 primarily. White arrows indicate VE-2B co-localized with both PSD-95 and SAP102.

    Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

    Techniques: Western Blot, Infection, Immunoprecipitation, Transfection

    VE-2B co-localized with PSD-95 from the TGN to the plasma membrane. (A) At 10 minutes after release from the ER, VE-2B showed little colocalization with antibody staining to endogenous PSD-95 in the soma (top panels; scale bars are 10 µm). Forty-five minutes after ER release (second row), a time point at which the leading edge of VE-2B reaches the surface, PSD-95 showed significant colocalization with VE-2B. Similar results were obtained with VE-2A (see Table S1 ). (B) Colocalization with PSD-95 was also evident in dendrites at 45 minutes and later time points, such as 3 hours (see Fig. 6). VE-2B (top panel) showed co-localization with endogenous PSD-95 (middle panel) in dendrites at 45 minutes after permissive temperature, and later (scale bar 5 µm). (C) Neurons transfected with VE-2B were subjected to 40°C then the medium was exchanged with medium equilibrated at 20°C. Cultures were maintained at 20°C for 1 hour to allow VE-2B cargo exiting the ER to build up in the TGN. Immunostaining for endogenous TGN38 (a TGN marker; top panels) and PSD-95 (second row) in VE-2B transfected neurons subjected to the 20°C temperature manipulation showed robust colocalization (scale bars 5 µm). (D) Quantification of PSD-95 co-localization at different stages of the secretory pathway. There was a significant difference among the groups using a one-way Anova. However, post hoc comparisons demonstrated an insignificant pairwise difference at 10 minutes following ER exit. PSD-95 showed levels of co-localization with VE-2B (open circles) not significantly different from those of VE-2BΔ7 (filled triangles) 10 minutes after ER release. Forty-five minutes after ER exit, VE-2B co-localization with PSD-95 (filled circles) was significantly enhanced at most thresholds when compared to VE-2B colocalization at 10 minutes after ER exit. The significant increase in colocalization of VE-2B with PSD-95 seen at 45 minutes after ER exit could be reproduced by switching media to 20°C for 1 hour (open squares). To verify that VE-2B was concentrated in the TGN after 1 hour 20°C incubation, colocalization of VE-2B with TGN38 was quantified (filled squares).

    Journal: PLoS ONE

    Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

    doi: 10.1371/journal.pone.0039585

    Figure Lengend Snippet: VE-2B co-localized with PSD-95 from the TGN to the plasma membrane. (A) At 10 minutes after release from the ER, VE-2B showed little colocalization with antibody staining to endogenous PSD-95 in the soma (top panels; scale bars are 10 µm). Forty-five minutes after ER release (second row), a time point at which the leading edge of VE-2B reaches the surface, PSD-95 showed significant colocalization with VE-2B. Similar results were obtained with VE-2A (see Table S1 ). (B) Colocalization with PSD-95 was also evident in dendrites at 45 minutes and later time points, such as 3 hours (see Fig. 6). VE-2B (top panel) showed co-localization with endogenous PSD-95 (middle panel) in dendrites at 45 minutes after permissive temperature, and later (scale bar 5 µm). (C) Neurons transfected with VE-2B were subjected to 40°C then the medium was exchanged with medium equilibrated at 20°C. Cultures were maintained at 20°C for 1 hour to allow VE-2B cargo exiting the ER to build up in the TGN. Immunostaining for endogenous TGN38 (a TGN marker; top panels) and PSD-95 (second row) in VE-2B transfected neurons subjected to the 20°C temperature manipulation showed robust colocalization (scale bars 5 µm). (D) Quantification of PSD-95 co-localization at different stages of the secretory pathway. There was a significant difference among the groups using a one-way Anova. However, post hoc comparisons demonstrated an insignificant pairwise difference at 10 minutes following ER exit. PSD-95 showed levels of co-localization with VE-2B (open circles) not significantly different from those of VE-2BΔ7 (filled triangles) 10 minutes after ER release. Forty-five minutes after ER exit, VE-2B co-localization with PSD-95 (filled circles) was significantly enhanced at most thresholds when compared to VE-2B colocalization at 10 minutes after ER exit. The significant increase in colocalization of VE-2B with PSD-95 seen at 45 minutes after ER exit could be reproduced by switching media to 20°C for 1 hour (open squares). To verify that VE-2B was concentrated in the TGN after 1 hour 20°C incubation, colocalization of VE-2B with TGN38 was quantified (filled squares).

    Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

    Techniques: Staining, Transfection, Immunostaining, Marker, Incubation

    VE-2B and the MAGUKs are consistent with co-transportation to the synapse. (A) VE-2B chimeras co-localized with high intensity PSD-95 (left graph) and SAP102 (right graph) in dendrites 3 hours after release with the red threshold fixed at 4X background (104–255 inclusive gray scale). Colocalization was quantified as described in Experimental Methods. VE, VE-2B, and VE-2BΔ7 colocalization was assessed across the range of inclusive thresholds for green indicated on the x-axis. There was a significant group effect by one-way Anova even at even the lowest green threshold (p

    Journal: PLoS ONE

    Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

    doi: 10.1371/journal.pone.0039585

    Figure Lengend Snippet: VE-2B and the MAGUKs are consistent with co-transportation to the synapse. (A) VE-2B chimeras co-localized with high intensity PSD-95 (left graph) and SAP102 (right graph) in dendrites 3 hours after release with the red threshold fixed at 4X background (104–255 inclusive gray scale). Colocalization was quantified as described in Experimental Methods. VE, VE-2B, and VE-2BΔ7 colocalization was assessed across the range of inclusive thresholds for green indicated on the x-axis. There was a significant group effect by one-way Anova even at even the lowest green threshold (p

    Article Snippet: Two separate anti-SAP102, and anti-PSD-95 antibodies have been characterized previously (rabbit anti-SAP102, JH62514; rabbit anti-SAP102, Alomone Labs; rabbit anti-PSD-95, T60; mouse PSD-95, Transduction Labs; .

    Techniques:

    PSD-95 expression pattern depends on culture duration. ( A , B ) Representative tdTomato-positive hRGCs ( red ) cultured for 1 ( A ) and 4 ( B ) weeks and immunolabeled against PSD-95 ( green ). Scale bar = 20 µm. ( C ) PSD-95 labeling within hRGC somas significantly decreased after 4 weeks versus earlier time points ( P ≤ 0.033). ( D ) Quantification of PSD-95 labeling within hRGC primary neurites. PSD-95 labeling significantly increased by 3 and 4 weeks in culture compared with 1-week cells. Significance indicators: * comparison with 1-week cells, # comparison with 2-week cells, ^ comparison with 3-week cells. Statistics: one-way analysis of variance, Tukey post hoc test ( C , D ). Data are mean ± standard error of the mean.

    Journal: Translational Vision Science & Technology

    Article Title: Intrinsic Morphologic and Physiologic Development of Human Derived Retinal Ganglion Cells In Vitro

    doi: 10.1167/tvst.10.10.1

    Figure Lengend Snippet: PSD-95 expression pattern depends on culture duration. ( A , B ) Representative tdTomato-positive hRGCs ( red ) cultured for 1 ( A ) and 4 ( B ) weeks and immunolabeled against PSD-95 ( green ). Scale bar = 20 µm. ( C ) PSD-95 labeling within hRGC somas significantly decreased after 4 weeks versus earlier time points ( P ≤ 0.033). ( D ) Quantification of PSD-95 labeling within hRGC primary neurites. PSD-95 labeling significantly increased by 3 and 4 weeks in culture compared with 1-week cells. Significance indicators: * comparison with 1-week cells, # comparison with 2-week cells, ^ comparison with 3-week cells. Statistics: one-way analysis of variance, Tukey post hoc test ( C , D ). Data are mean ± standard error of the mean.

    Article Snippet: After electrophysiologic recordings, hRGCs were fixed with 4% paraformaldehyde overnight at 4°C and immunolabeled with the following primary antibodies: RNA-binding protein with multiple splicing (RBPMS, GTX118619,1:200, Genetex, Irvine, CA) postsynaptic density 95 (PSD-95, APZ-009, 1:400, Alomone Labs, Jerusalem, Israel), and AnkG (338800, 1:250, Invitrogen, Carlsbad, CA).

    Techniques: Expressing, Cell Culture, Immunolabeling, Labeling