rabbit anti nrcam  (Developmental Studies Hybridoma Bank)

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Ankyrin B promotes developmental spine regulation in the mouse prefrontal cortex.

doi: 10.1093/cercor/bhad311

Figure Lengend Snippet: Fig. 3. Sema3F-induced spine retraction is impaired in cortical neuron cultures from Ank2-null, NrCAM-null, and NrCAM Y1231H mutant mice. (A) WT and Ank2-null cortical neuronal cultures were transfected with pCAG-IRES-EGFP, treated with 5 nM Fc or Sema3F-Fc for 30 min at DIV14, immunostained for EGFP, and apical dendrites imaged confocally (scale bar = 5 μm). Ank2-null cultures were co-transfected with Ank2-220 or Ank2-440 cDNAs. Representative images show EGFP-labeled apical dendrites with spines in WT and Ank2-null cortical neuronal cultures treated with Fc or Sema3F- Fc. Pial surface is toward the left. (B) Quantification of mean spine intensity ± SEM on apical dendrites of neuronal cultures described in (A). Each point represents the mean spine density per neuron per 10 μm dendrite length. Two-factor ANOVA with Tukey post hoc testing of spine density on Ank2-null neurons demonstrated that Ank2 deletion inhibited Sema3F-induced spine retraction (Fc-treated, 3.23 spines/10 μm ± 0.18; Sema3F-Fc treated, 3.01 spines/10 μm ± 0.15, P = 0.99). In contrast WT neurons exhibited spine retraction in response to Sema3F (Fc-treated, 3.24 spines/10 μm ± 0.14; Sema3F- Fc-treated, 2.45 spines/10 μm ± 0.11, ∗P < 0.001). Sema3F-Fc-induced spine retraction in Ank2-null neurons was rescued by re-expressing AnkB-220 cDNA (Fc-treated, 3.08 spines/10 μm ± 0.14; Sema3F-Fc-treated, 2.30 spines/10 μm ±0.10, ∗P = 0.001). AnkB-440 cDNA was not able to rescue Sema3F-induced spine retraction (Fc, treated, 3.59 spines/10 μm ± 0.20; Sema3F-Fc -treated, 3.43 spines/10 μm ±0.18, P = 0.99). Immunostaining with pan-AnkB antibodies verified equivalent levels of isoform expression (not shown). Points represent mean spine density of individual neurons. (C) WT cortical neuronal cultures were transfected with pCAG-IRES-EGFP, immunostained, and imaged confocally (DIV14). Representative confocal images of neurons with apical dendritic spines immunostained with pan-AnkB antibodies and Alexafluor-555 (red; A, D), GFP with Alexafluor-488 (green; B, E), and merged images (C, F). Control staining with secondary antibodies alone is shown in the upper right dotted inset in (A). Top panels are maximum intensity projections, with a single optical section of AnkB immunofluorescence staining of neurons shown in the lower right dotted inset in (A). AnkB immunofluorescence labeling was observed in spines with filopodial (fp), mushroom (mr), and stubby (st) morphology (D, arrows). At higher magnification AnkB immunolabeling (red) with Alexafluor-594 was evident within a spine head domain (arrows) by (G) confocal or (H) STED superresolution microscopy. Scale bars = 100 (A–C), 5 (D–F), 0.2 μm (G, H). (D) NrCAM-null cortical neuronal cultures were transfected with pCAG-IRES-EGFP vector alone, or plasmids containing WT or NrCAM Y1231H cDNAs. Cultures were treated with 5 nM Fc or Sema3F-Fc for 30 min at DIV14, immunostained for EGFP and apical dendrites imaged confocally (scale bar = 5 μm). Representative images of EGFP-labeled apical dendrites show that Sema3F-Fc promotes spine retraction only on NrCAM- null neurons re-expressing WT NrCAM. (E) Sema3F-Fc induces spine retraction on apical dendrites of NrCAM-null cortical neurons re-expressing WT NrCAM but not NrCAM Y1231H or empty vector. Two-factor ANOVA with Tukey post hoc testing showed a significant difference (∗P = 0.003) in spine density in neurons re-expressing WT NrCAM following treatment with control Fc (4.28 spines/10 μm ± 0.16) vs. Sema3F-Fc (3.28 spines/10 μm ± 0.15). In contrast, there was not a significant difference (P = 0.47) in spine density in neurons re-expressing NrCAM Y1231H following treatment with control Fc (4.25 spines/10 μm ± 0.22) vs. Sema3F-Fc (4.67 spines/10 μm ± 0.25). There was also not a significant difference (P > 0.99) in spine density in NrCAM-null neurons transfected with vector alone.

Article Snippet: Membranes were stripped and reprobed with rabbit anti-NrCAM antibodies (R&D Systems AF8538).

Techniques: Mutagenesis, Transfection, Labeling, Expressing, Immunostaining, Control, Staining, Immunofluorescence, Immunolabeling, Microscopy, Plasmid Preparation

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Ankyrin B promotes developmental spine regulation in the mouse prefrontal cortex.

doi: 10.1093/cercor/bhad311

Figure Lengend Snippet: Fig. 4. AnkB-220 associates with NrCAM at the FIGQY motif. (A) Representative immunoblotting (IB) of AnkB-220 (220 kDa) and NrCAM (130 kDa) in cortical lysates from postnatal mouse brain (P22-P105) (20 μg protein). Relative amounts of AnkB-220 or NrCAM were determined by densitometric scanning of respective protein bands. AnkB-220 and NrCAM expression were equivalent from P22-P105. Similar results were obtained in duplicate experiments. (B) Co-immunoprecipitation of AnkB-220 (220 kDa) and NrCAM (130 kDa) from equal amounts of protein (1 mg) in cortex lysates of postnatal mouse forebrain (P22-P105). NrCAM was immunoprecipitated (IP) and AnkB-220 detected by immunoblotting (IB). AnkB immunoblots (above) were reprobed for NrCAM (below). Densitometric scanning was performed and the ratio of immunoprecipitated AnkB-220 to NrCAM indicated below. (C) Co-immunoprecipitation of AnkB-220 with NrCAM from P28 mouse synaptoneurosomes, shown by immunoprecipitation (IP) with NrCAM antibodies or nonimmune IgG (NIg) and immunoblotting (IB) with AnkB antibodies. Blots were reprobed by immunoblotting with NrCAM antibodies (lower panels). Inputs represent synaptoneurosome lysates that were not subjected to immunoprecipitation. Example is representative of replicate blots. (D) Filtered homogenate (FH), first supernatant (S1), and synaptoneurosome (Syn) samples (equal protein, 25 μg) were blotted for Ankyrin B (AnkB). Membranes were stripped and reprobed for post-synaptic density protein 95 (PSD95, known synaptic protein), tubulin (a non-synaptic protein), and actin (loading control). AnkB and PSD95 showed enrichment in Syn fraction, whereas tubulin showed decreased expression in Syn fraction. (E) Co-immunoprecipitation of AnkB-220 with WT NrCAM or mutant NrCAM Y1231H from transfected HEK293T cells (equal amounts protein). NrCAM was immunoprecipiated from HEK293T cell lysates and immunoblotted for AnkB. Blots were reprobed for NrCAM (lower panels). Densitometric scanning of bands yielded ratios of AnkB-220 to NrCAM in the immunoprecipitated samples (below). Input lysates (equal protein) are shown at right. (F) Scheme of spine pruning initiated by Sema3F dimers. Sema3F binds the holoreceptor complex formed by NrCAM, Npn-2, and PlexA3. This binding event activates PlexinA3 Rap-GAP activity and subsequent downstream signaling leads to spine elimination via Rac1 and RhoA GTPase-governed pathways resulting in F-actin depolymerization. AnkB recruitment to the FIGQY motif in the NrCAM cytoplasmic domain may serve to stabilize the Sema3F complex, enhancing the signaling leading to spine pruning.

Article Snippet: Membranes were stripped and reprobed with rabbit anti-NrCAM antibodies (R&D Systems AF8538).

Techniques: Western Blot, Expressing, Immunoprecipitation, Control, Mutagenesis, Transfection, Binding Assay, Activity Assay