rabbit anti nl2  (Alomone Labs)

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

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

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

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