rabbit anti nl2  (Alomone Labs)


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    Name:
    Anti Neuroligin 2 extracellular Antibody
    Description:
    Anti Neuroligin 2 extracellular Antibody is directed against an extracellular epitope of rat Neuroligin 2 Anti Neuroligin 2 extracellular Antibody ANR 036 can be used in western blot immunohistochemistry and immunocytochemistry applications It has been designed to recognize NLGN2 from rat mouse and human samples
    Catalog Number:
    ANR-036
    Price:
    397.0
    Category:
    Primary Antibody
    Applications:
    Immunocytochemistry, Immunofluorescence, Immunohistochemistry, Live Cell Imaging, Western Blot
    Purity:
    Affinity purified on immobilized antigen.
    Immunogen:
    Synthetic peptide
    Size:
    25 mcl
    Antibody Type:
    Polyclonal Primary Antibodies
    Format:
    Lyophilized Powder
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
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    Structured Review

    Alomone Labs rabbit anti nl2
    Anti Neuroligin 2 extracellular Antibody
    Anti Neuroligin 2 extracellular Antibody is directed against an extracellular epitope of rat Neuroligin 2 Anti Neuroligin 2 extracellular Antibody ANR 036 can be used in western blot immunohistochemistry and immunocytochemistry applications It has been designed to recognize NLGN2 from rat mouse and human samples
    https://www.bioz.com/result/rabbit anti nl2/product/Alomone Labs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti nl2 - by Bioz Stars, 2021-09
    92/100 stars

    Images

    1) Product Images from "SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling"

    Article Title: SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2019.10.096

    Overexpression of SNX27 GFP WT but Not SNX27 GFP H112A Increases Postsynaptic Clusters and Inhibitory Signaling (A–C) Confocal images of 30 μm dendritic sections of hippocampal neurons, mock transfected (control) (A) or overexpressing SNX27 GFP WT (B) or SNX27 GFP H112A (C). Neurons were stained for GAD65, total NL2 (NL2 TTL ), synaptic NL2 (NL2 EXT ), gephyrin, or the GABA A R γ2 subunit. Arrowheads show synaptic clusters. Dashed lines indicate the dendritic outline for mock-transfected cells (A). Scale bar, 4 μm. (D–H) Quantification of cluster number (left) and area (right) in hippocampal neurons either mock transfected (Ctrl) or overexpressing SNX27 GFP WT (WT) or SNX27 GFP H112A (H112A). Quantified are NL2 TTL (D) (n = 21, 19, and 19), NL2 EXT (E) (n = 24, 23, and 24), γ2 (F) (n = 35, 46, and 52), gephyrin (G) (n = 25, 34, and 26), and GAD65 (H) (n = 21, 20, and 17). (I) Representative traces of mIPSC patch-clamp recordings from hippocampal cultures, mock transfected (Control) or overexpressing SNX27 GFP WT. (J and K) Pooled data (left) and cumulative probability plot (right) of mIPSCs amplitude (J) and frequency (K) (n = 13 and 22). Values are mean ± SEM. ∗ p
    Figure Legend Snippet: Overexpression of SNX27 GFP WT but Not SNX27 GFP H112A Increases Postsynaptic Clusters and Inhibitory Signaling (A–C) Confocal images of 30 μm dendritic sections of hippocampal neurons, mock transfected (control) (A) or overexpressing SNX27 GFP WT (B) or SNX27 GFP H112A (C). Neurons were stained for GAD65, total NL2 (NL2 TTL ), synaptic NL2 (NL2 EXT ), gephyrin, or the GABA A R γ2 subunit. Arrowheads show synaptic clusters. Dashed lines indicate the dendritic outline for mock-transfected cells (A). Scale bar, 4 μm. (D–H) Quantification of cluster number (left) and area (right) in hippocampal neurons either mock transfected (Ctrl) or overexpressing SNX27 GFP WT (WT) or SNX27 GFP H112A (H112A). Quantified are NL2 TTL (D) (n = 21, 19, and 19), NL2 EXT (E) (n = 24, 23, and 24), γ2 (F) (n = 35, 46, and 52), gephyrin (G) (n = 25, 34, and 26), and GAD65 (H) (n = 21, 20, and 17). (I) Representative traces of mIPSC patch-clamp recordings from hippocampal cultures, mock transfected (Control) or overexpressing SNX27 GFP WT. (J and K) Pooled data (left) and cumulative probability plot (right) of mIPSCs amplitude (J) and frequency (K) (n = 13 and 22). Values are mean ± SEM. ∗ p

    Techniques Used: Over Expression, Transfection, Staining, Patch Clamp

    SNX27 Knockdown Decreases Synaptic NL2 and Disrupts Inhibitory Signaling (A and B) Confocal images of 30 μm dendritic sections of hippocampal neurons, overexpressing control shRNAi (A) or SNX27-specific shRNAi (B). Neurons were stained as in Figures 3 A–3C. Arrowheads show synaptic clusters. Scale bar, 4 μm. (C–G) Quantification of cluster number (left) and area (right) in hippocampal neurons transfected as in (A) and (B). Quantified are NL2 TTL (C) (n = 19 and 17; cluster number, unpaired two-tailed t test; cluster area, Mann-Whitney test), NL2 EXT (D) (n = 36 and 37; Mann-Whitney tests), γ2 (E) (n = 20 and 19; unpaired two-tailed t tests), gephyrin (F) (n = 19 and 17; Mann-Whitney tests), and GAD65 (G) (n = 27; cluster number, Mann-Whitney test; cluster area, unpaired two-tailed t test). (H) Representative traces of mIPSC patch-clamp recordings from hippocampal cultures overexpressing control or SNX27-specific shRNAi. (I and J) Pooled data (left) and cumulative probability (right) of mIPSC amplitude (I) and frequency (J) (n = 23 and 20; unpaired one-tailed t tests). (K and L) Quantification of cluster number (left) and area (right) in hippocampal neurons transfected with SNX27-specific shRNAi alone (red) or combined with RNAi-resistant SNX27 GFP WT (black) or SNX27 GFP H112A (gray). Quantified are NL2 EXT (K) (n = 22, 23, and 19; one-way ANOVA with Bonferroni’s correction) and GAD65 (L) (n = 23, 22, and 28; Kruskal-Wallis test with Dunn’s correction). See Figures S4 I and S4J for quantification of γ2 and gephyrin. (M) Representative traces of mIPSC patch-clamp recordings from hippocampal cultures overexpressing SNX27-specific shRNAi alone (KD) or combined with RNAi-resistant SNX27 GFP WT (KD+WT) or SNX27 GFP H112A (KD+H112A). (N and O) Pooled data (left) and cumulative probability (right) of mIPSC amplitude (N) and frequency (O) (n = 14, 14, and 13; unpaired one-tailed t tests). (P) Quantification of mIPSC charge transfer (unpaired one-tailed t tests). Values are mean ± SEM. ∗ p
    Figure Legend Snippet: SNX27 Knockdown Decreases Synaptic NL2 and Disrupts Inhibitory Signaling (A and B) Confocal images of 30 μm dendritic sections of hippocampal neurons, overexpressing control shRNAi (A) or SNX27-specific shRNAi (B). Neurons were stained as in Figures 3 A–3C. Arrowheads show synaptic clusters. Scale bar, 4 μm. (C–G) Quantification of cluster number (left) and area (right) in hippocampal neurons transfected as in (A) and (B). Quantified are NL2 TTL (C) (n = 19 and 17; cluster number, unpaired two-tailed t test; cluster area, Mann-Whitney test), NL2 EXT (D) (n = 36 and 37; Mann-Whitney tests), γ2 (E) (n = 20 and 19; unpaired two-tailed t tests), gephyrin (F) (n = 19 and 17; Mann-Whitney tests), and GAD65 (G) (n = 27; cluster number, Mann-Whitney test; cluster area, unpaired two-tailed t test). (H) Representative traces of mIPSC patch-clamp recordings from hippocampal cultures overexpressing control or SNX27-specific shRNAi. (I and J) Pooled data (left) and cumulative probability (right) of mIPSC amplitude (I) and frequency (J) (n = 23 and 20; unpaired one-tailed t tests). (K and L) Quantification of cluster number (left) and area (right) in hippocampal neurons transfected with SNX27-specific shRNAi alone (red) or combined with RNAi-resistant SNX27 GFP WT (black) or SNX27 GFP H112A (gray). Quantified are NL2 EXT (K) (n = 22, 23, and 19; one-way ANOVA with Bonferroni’s correction) and GAD65 (L) (n = 23, 22, and 28; Kruskal-Wallis test with Dunn’s correction). See Figures S4 I and S4J for quantification of γ2 and gephyrin. (M) Representative traces of mIPSC patch-clamp recordings from hippocampal cultures overexpressing SNX27-specific shRNAi alone (KD) or combined with RNAi-resistant SNX27 GFP WT (KD+WT) or SNX27 GFP H112A (KD+H112A). (N and O) Pooled data (left) and cumulative probability (right) of mIPSC amplitude (N) and frequency (O) (n = 14, 14, and 13; unpaired one-tailed t tests). (P) Quantification of mIPSC charge transfer (unpaired one-tailed t tests). Values are mean ± SEM. ∗ p

    Techniques Used: Staining, Transfection, Two Tailed Test, MANN-WHITNEY, Patch Clamp, One-tailed Test

    Internalized NL2 Co-localizes and Interacts with SNX27 and Retromer (A and B) Confocal images of antibody feeding in hippocampal neurons co-expressing HA NL2 with either SNX27 GFP (A) or VPS35 GFP (B). Arrowheads show examples of co-localization. Scale bars, 25 μm (whole cell) and 5 μm (soma). (C–F) Western blots of co-immunoprecipitation from rat brain lysate via endogenous NL2 (C–E) showing interaction with endogenous SNX27 (C), VPS35 (D), and VPS26 (E) or via endogenous SNX27 (F) showing interaction with endogenous NL2 and VPS35. IP, immunoprecipitation. Numbers on the left indicate molecular weight in kDa. (G) Western blot of GST pull-down from rat brain lysate. See also Figure S1 .
    Figure Legend Snippet: Internalized NL2 Co-localizes and Interacts with SNX27 and Retromer (A and B) Confocal images of antibody feeding in hippocampal neurons co-expressing HA NL2 with either SNX27 GFP (A) or VPS35 GFP (B). Arrowheads show examples of co-localization. Scale bars, 25 μm (whole cell) and 5 μm (soma). (C–F) Western blots of co-immunoprecipitation from rat brain lysate via endogenous NL2 (C–E) showing interaction with endogenous SNX27 (C), VPS35 (D), and VPS26 (E) or via endogenous SNX27 (F) showing interaction with endogenous NL2 and VPS35. IP, immunoprecipitation. Numbers on the left indicate molecular weight in kDa. (G) Western blot of GST pull-down from rat brain lysate. See also Figure S1 .

    Techniques Used: Expressing, Western Blot, Immunoprecipitation, Molecular Weight

    SNX27 Regulates Recycling of NL2 via PDZ-Ligand Interaction (A) Schematic representation of SNX27 (left) and NL2 (right), indicating the positions of mutations generated for this study. F1-3, FERM1-3; PDZL, PDZ-ligand; TM, transmembrane. (B and C) Western blots of co-immunoprecipitation from COS-7 cells co-expressing either WT HA NL2 with WT or mutant SNX27 GFP (B) or WT SNX27 GFP with WT or mutant HA NL2 (C). SNX27 GFP was pulled down using GFP-Trap beads. IP, immunoprecipitation. (D) Confocal images of antibody feeding in HeLa cells co-expressing HA NL2 WT or ΔPDZL with SNX27 GFP . Scale bars, 10 μm (whole cell) and 3 μm (zooms). Arrowheads show examples of co-localization. (E) Confocal images of antibody feeding in HeLa cells expressing HA NL2 WT or ΔPDZL. Scale bar, 10 μm. (F) Quantification of relative HA NL2 internalization in HeLa cells (n = 16; unpaired two-tailed t test). (G) Confocal images of antibody recycling in HeLa cells expressing HA NL2 WT or ΔPDZL. To enable better visualization of extracellular HA NL2 upon recycling, only the channel representing surface labeling is shown after thresholding and binarizing. Dashed lines indicate the outlines of the cell. Scale bar, 10 μm. (H) Quantification of relative HA NL2 recycling in HeLa cells (n = 27; one-way ANOVA with Bonferroni’s correction). Values are mean ± SEM; n.s., non-significant. ∗∗ p
    Figure Legend Snippet: SNX27 Regulates Recycling of NL2 via PDZ-Ligand Interaction (A) Schematic representation of SNX27 (left) and NL2 (right), indicating the positions of mutations generated for this study. F1-3, FERM1-3; PDZL, PDZ-ligand; TM, transmembrane. (B and C) Western blots of co-immunoprecipitation from COS-7 cells co-expressing either WT HA NL2 with WT or mutant SNX27 GFP (B) or WT SNX27 GFP with WT or mutant HA NL2 (C). SNX27 GFP was pulled down using GFP-Trap beads. IP, immunoprecipitation. (D) Confocal images of antibody feeding in HeLa cells co-expressing HA NL2 WT or ΔPDZL with SNX27 GFP . Scale bars, 10 μm (whole cell) and 3 μm (zooms). Arrowheads show examples of co-localization. (E) Confocal images of antibody feeding in HeLa cells expressing HA NL2 WT or ΔPDZL. Scale bar, 10 μm. (F) Quantification of relative HA NL2 internalization in HeLa cells (n = 16; unpaired two-tailed t test). (G) Confocal images of antibody recycling in HeLa cells expressing HA NL2 WT or ΔPDZL. To enable better visualization of extracellular HA NL2 upon recycling, only the channel representing surface labeling is shown after thresholding and binarizing. Dashed lines indicate the outlines of the cell. Scale bar, 10 μm. (H) Quantification of relative HA NL2 recycling in HeLa cells (n = 27; one-way ANOVA with Bonferroni’s correction). Values are mean ± SEM; n.s., non-significant. ∗∗ p

    Techniques Used: Generated, Western Blot, Immunoprecipitation, Expressing, Mutagenesis, Two Tailed Test, Labeling

    Related Articles

    Incubation:

    Article Title: Differential regulation of synaptic and extrasynaptic α4 GABA(A) receptor populations by protein kinase A and protein kinase C in cultured cortical neurons
    Article Snippet: .. 30–50μg of protein was electrophoresed on 4–16% Tris-Glycine polyacrylamide gels (Biorad) and transferred to iBlot PVDF membranes (Invitrogen), blocked for 1 h in 1–5% BSA and incubated overnight at 4 °C with either anti-GABAA α4 (Abcam, #ab117080, 1:500), anti-GABAA β2 (Novus, #NB300-198, 1:1000), anti-GABAA β3 (Novus, #NBP1-47613, 1:1000), anti-GABAA δ (Novus, #3002-200, 1:750), anti-GABAA γ2 (Novus, #NB300-190, 1:1000), anti-GABAA phospho-γ2 (Ser327 )(PhosphoSolutions, p1130-327, 1:1000), anti-GABAA phosphor-β3 (Novus, NBP2-29508, 1:1000) anti-PSD95 (Novus, #NB300-198, 1:2000), anti-Gephyrin (BD Transduction, #610584, 1:1000), anti-neuroligin2 (Alomone Labs, ANR-036, 1:1000) or β-actin (Novus, #NB600-501, 1:3000) Membranes were then incubated with peroxidase-labeled secondary antibodies (Mouse, rabbit, goat, Jackson Laboratories, 1:10000) and signals were developed using ECLPrime (GE) on a LAS 4000 Imager (GE). ..

    Article Title: SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling
    Article Snippet: .. For surface labeling, cells were incubated in block solution (PBS, 10% horse serum, 0.5% BSA) for 10 mins, and then stained for 1h at RT with primary antibody diluted in block solution (1:100 Rabbit-anti-NL2 (Alomone Labs, ANR-036), 1:500 Guinea Pig-anti-γ2 (Synaptic Systems, 224-004)). ..

    Article Title: SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling
    Article Snippet: .. For co-immunoprecipitations from brain lysate, 4 mg of brain lysate was incubated overnight at 4°C with rotation with 1 μg Rabbit IgG (ThermoFisher, 10500C) or Rabbit-anti-NL2 (Alomone Labs, ANR-036), or with 5 μg Mouse IgG (ThermoFisher, 10400C) or Mouse-anti-SNX27 (Abcam, ab77799). ..

    Labeling:

    Article Title: SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling
    Article Snippet: .. For surface labeling, cells were incubated in block solution (PBS, 10% horse serum, 0.5% BSA) for 10 mins, and then stained for 1h at RT with primary antibody diluted in block solution (1:100 Rabbit-anti-NL2 (Alomone Labs, ANR-036), 1:500 Guinea Pig-anti-γ2 (Synaptic Systems, 224-004)). ..

    Blocking Assay:

    Article Title: SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling
    Article Snippet: .. For surface labeling, cells were incubated in block solution (PBS, 10% horse serum, 0.5% BSA) for 10 mins, and then stained for 1h at RT with primary antibody diluted in block solution (1:100 Rabbit-anti-NL2 (Alomone Labs, ANR-036), 1:500 Guinea Pig-anti-γ2 (Synaptic Systems, 224-004)). ..

    Staining:

    Article Title: SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling
    Article Snippet: .. For surface labeling, cells were incubated in block solution (PBS, 10% horse serum, 0.5% BSA) for 10 mins, and then stained for 1h at RT with primary antibody diluted in block solution (1:100 Rabbit-anti-NL2 (Alomone Labs, ANR-036), 1:500 Guinea Pig-anti-γ2 (Synaptic Systems, 224-004)). ..

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    Alomone Labs rabbit anti nl2
    Overexpression of SNX27 GFP WT but Not SNX27 GFP H112A Increases Postsynaptic Clusters and Inhibitory Signaling (A–C) Confocal images of 30 μm dendritic sections of hippocampal neurons, mock transfected (control) (A) or overexpressing SNX27 GFP WT (B) or SNX27 GFP H112A (C). Neurons were stained for GAD65, total <t>NL2</t> (NL2 TTL ), synaptic NL2 (NL2 EXT ), gephyrin, or the GABA A R γ2 subunit. Arrowheads show synaptic clusters. Dashed lines indicate the dendritic outline for mock-transfected cells (A). Scale bar, 4 μm. (D–H) Quantification of cluster number (left) and area (right) in hippocampal neurons either mock transfected (Ctrl) or overexpressing SNX27 GFP WT (WT) or SNX27 GFP H112A (H112A). Quantified are NL2 TTL (D) (n = 21, 19, and 19), NL2 EXT (E) (n = 24, 23, and 24), γ2 (F) (n = 35, 46, and 52), gephyrin (G) (n = 25, 34, and 26), and GAD65 (H) (n = 21, 20, and 17). (I) Representative traces of mIPSC patch-clamp recordings from hippocampal cultures, mock transfected (Control) or overexpressing SNX27 GFP WT. (J and K) Pooled data (left) and cumulative probability plot (right) of mIPSCs amplitude (J) and frequency (K) (n = 13 and 22). Values are mean ± SEM. ∗ p
    Rabbit Anti Nl2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti nl2/product/Alomone Labs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti nl2 - by Bioz Stars, 2021-09
    92/100 stars
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    Image Search Results


    Overexpression of SNX27 GFP WT but Not SNX27 GFP H112A Increases Postsynaptic Clusters and Inhibitory Signaling (A–C) Confocal images of 30 μm dendritic sections of hippocampal neurons, mock transfected (control) (A) or overexpressing SNX27 GFP WT (B) or SNX27 GFP H112A (C). Neurons were stained for GAD65, total NL2 (NL2 TTL ), synaptic NL2 (NL2 EXT ), gephyrin, or the GABA A R γ2 subunit. Arrowheads show synaptic clusters. Dashed lines indicate the dendritic outline for mock-transfected cells (A). Scale bar, 4 μm. (D–H) Quantification of cluster number (left) and area (right) in hippocampal neurons either mock transfected (Ctrl) or overexpressing SNX27 GFP WT (WT) or SNX27 GFP H112A (H112A). Quantified are NL2 TTL (D) (n = 21, 19, and 19), NL2 EXT (E) (n = 24, 23, and 24), γ2 (F) (n = 35, 46, and 52), gephyrin (G) (n = 25, 34, and 26), and GAD65 (H) (n = 21, 20, and 17). (I) Representative traces of mIPSC patch-clamp recordings from hippocampal cultures, mock transfected (Control) or overexpressing SNX27 GFP WT. (J and K) Pooled data (left) and cumulative probability plot (right) of mIPSCs amplitude (J) and frequency (K) (n = 13 and 22). Values are mean ± SEM. ∗ p

    Journal: Cell Reports

    Article Title: SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling

    doi: 10.1016/j.celrep.2019.10.096

    Figure Lengend Snippet: Overexpression of SNX27 GFP WT but Not SNX27 GFP H112A Increases Postsynaptic Clusters and Inhibitory Signaling (A–C) Confocal images of 30 μm dendritic sections of hippocampal neurons, mock transfected (control) (A) or overexpressing SNX27 GFP WT (B) or SNX27 GFP H112A (C). Neurons were stained for GAD65, total NL2 (NL2 TTL ), synaptic NL2 (NL2 EXT ), gephyrin, or the GABA A R γ2 subunit. Arrowheads show synaptic clusters. Dashed lines indicate the dendritic outline for mock-transfected cells (A). Scale bar, 4 μm. (D–H) Quantification of cluster number (left) and area (right) in hippocampal neurons either mock transfected (Ctrl) or overexpressing SNX27 GFP WT (WT) or SNX27 GFP H112A (H112A). Quantified are NL2 TTL (D) (n = 21, 19, and 19), NL2 EXT (E) (n = 24, 23, and 24), γ2 (F) (n = 35, 46, and 52), gephyrin (G) (n = 25, 34, and 26), and GAD65 (H) (n = 21, 20, and 17). (I) Representative traces of mIPSC patch-clamp recordings from hippocampal cultures, mock transfected (Control) or overexpressing SNX27 GFP WT. (J and K) Pooled data (left) and cumulative probability plot (right) of mIPSCs amplitude (J) and frequency (K) (n = 13 and 22). Values are mean ± SEM. ∗ p

    Article Snippet: For co-immunoprecipitations from brain lysate, 4 mg of brain lysate was incubated overnight at 4°C with rotation with 1 μg Rabbit IgG (ThermoFisher, 10500C) or Rabbit-anti-NL2 (Alomone Labs, ANR-036), or with 5 μg Mouse IgG (ThermoFisher, 10400C) or Mouse-anti-SNX27 (Abcam, ab77799).

    Techniques: Over Expression, Transfection, Staining, Patch Clamp

    SNX27 Knockdown Decreases Synaptic NL2 and Disrupts Inhibitory Signaling (A and B) Confocal images of 30 μm dendritic sections of hippocampal neurons, overexpressing control shRNAi (A) or SNX27-specific shRNAi (B). Neurons were stained as in Figures 3 A–3C. Arrowheads show synaptic clusters. Scale bar, 4 μm. (C–G) Quantification of cluster number (left) and area (right) in hippocampal neurons transfected as in (A) and (B). Quantified are NL2 TTL (C) (n = 19 and 17; cluster number, unpaired two-tailed t test; cluster area, Mann-Whitney test), NL2 EXT (D) (n = 36 and 37; Mann-Whitney tests), γ2 (E) (n = 20 and 19; unpaired two-tailed t tests), gephyrin (F) (n = 19 and 17; Mann-Whitney tests), and GAD65 (G) (n = 27; cluster number, Mann-Whitney test; cluster area, unpaired two-tailed t test). (H) Representative traces of mIPSC patch-clamp recordings from hippocampal cultures overexpressing control or SNX27-specific shRNAi. (I and J) Pooled data (left) and cumulative probability (right) of mIPSC amplitude (I) and frequency (J) (n = 23 and 20; unpaired one-tailed t tests). (K and L) Quantification of cluster number (left) and area (right) in hippocampal neurons transfected with SNX27-specific shRNAi alone (red) or combined with RNAi-resistant SNX27 GFP WT (black) or SNX27 GFP H112A (gray). Quantified are NL2 EXT (K) (n = 22, 23, and 19; one-way ANOVA with Bonferroni’s correction) and GAD65 (L) (n = 23, 22, and 28; Kruskal-Wallis test with Dunn’s correction). See Figures S4 I and S4J for quantification of γ2 and gephyrin. (M) Representative traces of mIPSC patch-clamp recordings from hippocampal cultures overexpressing SNX27-specific shRNAi alone (KD) or combined with RNAi-resistant SNX27 GFP WT (KD+WT) or SNX27 GFP H112A (KD+H112A). (N and O) Pooled data (left) and cumulative probability (right) of mIPSC amplitude (N) and frequency (O) (n = 14, 14, and 13; unpaired one-tailed t tests). (P) Quantification of mIPSC charge transfer (unpaired one-tailed t tests). Values are mean ± SEM. ∗ p

    Journal: Cell Reports

    Article Title: SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling

    doi: 10.1016/j.celrep.2019.10.096

    Figure Lengend Snippet: SNX27 Knockdown Decreases Synaptic NL2 and Disrupts Inhibitory Signaling (A and B) Confocal images of 30 μm dendritic sections of hippocampal neurons, overexpressing control shRNAi (A) or SNX27-specific shRNAi (B). Neurons were stained as in Figures 3 A–3C. Arrowheads show synaptic clusters. Scale bar, 4 μm. (C–G) Quantification of cluster number (left) and area (right) in hippocampal neurons transfected as in (A) and (B). Quantified are NL2 TTL (C) (n = 19 and 17; cluster number, unpaired two-tailed t test; cluster area, Mann-Whitney test), NL2 EXT (D) (n = 36 and 37; Mann-Whitney tests), γ2 (E) (n = 20 and 19; unpaired two-tailed t tests), gephyrin (F) (n = 19 and 17; Mann-Whitney tests), and GAD65 (G) (n = 27; cluster number, Mann-Whitney test; cluster area, unpaired two-tailed t test). (H) Representative traces of mIPSC patch-clamp recordings from hippocampal cultures overexpressing control or SNX27-specific shRNAi. (I and J) Pooled data (left) and cumulative probability (right) of mIPSC amplitude (I) and frequency (J) (n = 23 and 20; unpaired one-tailed t tests). (K and L) Quantification of cluster number (left) and area (right) in hippocampal neurons transfected with SNX27-specific shRNAi alone (red) or combined with RNAi-resistant SNX27 GFP WT (black) or SNX27 GFP H112A (gray). Quantified are NL2 EXT (K) (n = 22, 23, and 19; one-way ANOVA with Bonferroni’s correction) and GAD65 (L) (n = 23, 22, and 28; Kruskal-Wallis test with Dunn’s correction). See Figures S4 I and S4J for quantification of γ2 and gephyrin. (M) Representative traces of mIPSC patch-clamp recordings from hippocampal cultures overexpressing SNX27-specific shRNAi alone (KD) or combined with RNAi-resistant SNX27 GFP WT (KD+WT) or SNX27 GFP H112A (KD+H112A). (N and O) Pooled data (left) and cumulative probability (right) of mIPSC amplitude (N) and frequency (O) (n = 14, 14, and 13; unpaired one-tailed t tests). (P) Quantification of mIPSC charge transfer (unpaired one-tailed t tests). Values are mean ± SEM. ∗ p

    Article Snippet: For co-immunoprecipitations from brain lysate, 4 mg of brain lysate was incubated overnight at 4°C with rotation with 1 μg Rabbit IgG (ThermoFisher, 10500C) or Rabbit-anti-NL2 (Alomone Labs, ANR-036), or with 5 μg Mouse IgG (ThermoFisher, 10400C) or Mouse-anti-SNX27 (Abcam, ab77799).

    Techniques: Staining, Transfection, Two Tailed Test, MANN-WHITNEY, Patch Clamp, One-tailed Test

    Internalized NL2 Co-localizes and Interacts with SNX27 and Retromer (A and B) Confocal images of antibody feeding in hippocampal neurons co-expressing HA NL2 with either SNX27 GFP (A) or VPS35 GFP (B). Arrowheads show examples of co-localization. Scale bars, 25 μm (whole cell) and 5 μm (soma). (C–F) Western blots of co-immunoprecipitation from rat brain lysate via endogenous NL2 (C–E) showing interaction with endogenous SNX27 (C), VPS35 (D), and VPS26 (E) or via endogenous SNX27 (F) showing interaction with endogenous NL2 and VPS35. IP, immunoprecipitation. Numbers on the left indicate molecular weight in kDa. (G) Western blot of GST pull-down from rat brain lysate. See also Figure S1 .

    Journal: Cell Reports

    Article Title: SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling

    doi: 10.1016/j.celrep.2019.10.096

    Figure Lengend Snippet: Internalized NL2 Co-localizes and Interacts with SNX27 and Retromer (A and B) Confocal images of antibody feeding in hippocampal neurons co-expressing HA NL2 with either SNX27 GFP (A) or VPS35 GFP (B). Arrowheads show examples of co-localization. Scale bars, 25 μm (whole cell) and 5 μm (soma). (C–F) Western blots of co-immunoprecipitation from rat brain lysate via endogenous NL2 (C–E) showing interaction with endogenous SNX27 (C), VPS35 (D), and VPS26 (E) or via endogenous SNX27 (F) showing interaction with endogenous NL2 and VPS35. IP, immunoprecipitation. Numbers on the left indicate molecular weight in kDa. (G) Western blot of GST pull-down from rat brain lysate. See also Figure S1 .

    Article Snippet: For co-immunoprecipitations from brain lysate, 4 mg of brain lysate was incubated overnight at 4°C with rotation with 1 μg Rabbit IgG (ThermoFisher, 10500C) or Rabbit-anti-NL2 (Alomone Labs, ANR-036), or with 5 μg Mouse IgG (ThermoFisher, 10400C) or Mouse-anti-SNX27 (Abcam, ab77799).

    Techniques: Expressing, Western Blot, Immunoprecipitation, Molecular Weight

    SNX27 Regulates Recycling of NL2 via PDZ-Ligand Interaction (A) Schematic representation of SNX27 (left) and NL2 (right), indicating the positions of mutations generated for this study. F1-3, FERM1-3; PDZL, PDZ-ligand; TM, transmembrane. (B and C) Western blots of co-immunoprecipitation from COS-7 cells co-expressing either WT HA NL2 with WT or mutant SNX27 GFP (B) or WT SNX27 GFP with WT or mutant HA NL2 (C). SNX27 GFP was pulled down using GFP-Trap beads. IP, immunoprecipitation. (D) Confocal images of antibody feeding in HeLa cells co-expressing HA NL2 WT or ΔPDZL with SNX27 GFP . Scale bars, 10 μm (whole cell) and 3 μm (zooms). Arrowheads show examples of co-localization. (E) Confocal images of antibody feeding in HeLa cells expressing HA NL2 WT or ΔPDZL. Scale bar, 10 μm. (F) Quantification of relative HA NL2 internalization in HeLa cells (n = 16; unpaired two-tailed t test). (G) Confocal images of antibody recycling in HeLa cells expressing HA NL2 WT or ΔPDZL. To enable better visualization of extracellular HA NL2 upon recycling, only the channel representing surface labeling is shown after thresholding and binarizing. Dashed lines indicate the outlines of the cell. Scale bar, 10 μm. (H) Quantification of relative HA NL2 recycling in HeLa cells (n = 27; one-way ANOVA with Bonferroni’s correction). Values are mean ± SEM; n.s., non-significant. ∗∗ p

    Journal: Cell Reports

    Article Title: SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling

    doi: 10.1016/j.celrep.2019.10.096

    Figure Lengend Snippet: SNX27 Regulates Recycling of NL2 via PDZ-Ligand Interaction (A) Schematic representation of SNX27 (left) and NL2 (right), indicating the positions of mutations generated for this study. F1-3, FERM1-3; PDZL, PDZ-ligand; TM, transmembrane. (B and C) Western blots of co-immunoprecipitation from COS-7 cells co-expressing either WT HA NL2 with WT or mutant SNX27 GFP (B) or WT SNX27 GFP with WT or mutant HA NL2 (C). SNX27 GFP was pulled down using GFP-Trap beads. IP, immunoprecipitation. (D) Confocal images of antibody feeding in HeLa cells co-expressing HA NL2 WT or ΔPDZL with SNX27 GFP . Scale bars, 10 μm (whole cell) and 3 μm (zooms). Arrowheads show examples of co-localization. (E) Confocal images of antibody feeding in HeLa cells expressing HA NL2 WT or ΔPDZL. Scale bar, 10 μm. (F) Quantification of relative HA NL2 internalization in HeLa cells (n = 16; unpaired two-tailed t test). (G) Confocal images of antibody recycling in HeLa cells expressing HA NL2 WT or ΔPDZL. To enable better visualization of extracellular HA NL2 upon recycling, only the channel representing surface labeling is shown after thresholding and binarizing. Dashed lines indicate the outlines of the cell. Scale bar, 10 μm. (H) Quantification of relative HA NL2 recycling in HeLa cells (n = 27; one-way ANOVA with Bonferroni’s correction). Values are mean ± SEM; n.s., non-significant. ∗∗ p

    Article Snippet: For co-immunoprecipitations from brain lysate, 4 mg of brain lysate was incubated overnight at 4°C with rotation with 1 μg Rabbit IgG (ThermoFisher, 10500C) or Rabbit-anti-NL2 (Alomone Labs, ANR-036), or with 5 μg Mouse IgG (ThermoFisher, 10400C) or Mouse-anti-SNX27 (Abcam, ab77799).

    Techniques: Generated, Western Blot, Immunoprecipitation, Expressing, Mutagenesis, Two Tailed Test, Labeling