Journal: eLife

Article Title: Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A

doi: 10.7554/eLife.69815

Figure Lengend Snippet: ( A ) Western blot analysis of the expression of Sema 3 A, L1CAM and the Sema 3 A receptors Nrp1 and Plexin A1 in the cortex of PND1 control and AnkB440 KO mice. ( B ) Quantification of protein levels normalized to actin in cortical lysates from PND1 mice of indicated genotypes relative to the normalized levels of each protein in control brains. Data show mean ± SEM for three biological replicates per genotype for one experiment. Unpaired t test. *p < 0.05. ( C ) Western blot analysis of total and surface levels of indicated proteins in DIV3 control and AnkB440 KO cortical neurons. ( D ) Quantification of total and surface protein levels normalized to actin relative to the normalized levels of each protein in control cortical neurons. Data show mean ± SEM for three biological replicates per genotype for one experiment. Unpaired t test. **p < 0.01. ( E ) Images show PLA signal between L1CAM and Nrp1 at the cell surface of the axon and GCs of DIV3 cortical neurons from the indicated genotypes. This assay used an Nrp1 antibody that selectively recognizes an extracellular epitope. Phalloidin staining was used to identify GCs. Scale bar, 2 μm. ( F ) Quantification of PLA signal at the GC surface relative to GC area collected from an average n = 15 GCs/genotype. The box and whisker plots represent all data points arranged from minimum to maximum. One-way ANOVA with Tukey’s post hoc analysis test for multiple comparisons. ****p < 0.0001. ( G ) Images show Nrp1 localization at the surface of GCs of DIV3 neurons untreated and treated with Sema 3 A, which induces the internalization of surface Nrp1. Scale bar, 1 μm. ( H ) Quantification of surface Nrp1 levels at GCs relative to GC area at the basal state and upon Sema 3A-induced Nrp1 internalization. Data represent mean ± SEM collected from an average of n = 90 GCs/treatment/genotype/experiment. Each dot represents one out of three independent experiments. One-way ANOVA with Tukey’s post hoc analysis test for multiple comparisons. **p < 0.01. Figure 7—source data 1. AnkB440 stabilizes the L1CAM-Nrp1 complex at the cell surface of GCs.

Article Snippet: We also used rabbit anti-Plexin A1 (1:200, # APR-081) from Alomone; mouse anti-GluR1 (1:500, clone N355/1, #75–327) from Antibodies Incorporated; rabbit anti-phospho-Cofilin (Ser3) (1:1,000, #3311), rabbit anti-LIMK1 (1:1,000, #3842), rabbit anti-MAG (1:1,000, clone D4G3, #9043), rabbit anti-MOG (1:1,000, clone E5K6T, #96457), and mouse anti-GFAP (1:1,000, clone GA5, #3670) from Cell Signaling; mouse anti-cofilin (1:1,000, clone 1G6A2, #66057–1-Ig) from Proteintech; mouse anti-actin (1:2,000, clone C4, #MAB1501), and mouse anti-CNPase (1:500, #C5922) from Millipore-Sigma and rabbit anti-myelin PLP (1:1,000, #NBP1-87781) from Novus Biologicals.

Techniques: Western Blot, Expressing, Staining, Whisker Assay

Journal: eLife

Article Title: Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A

doi: 10.7554/eLife.69815

Figure Lengend Snippet: ( A ) Western blot analysis of the expression of Sema 3 A, L1CAM and the Sema 3 A receptors Nrp1 and Plexin A1 in the cortex of PND1 control and AnkB440 KO mice. ( B ) Quantification of protein levels normalized to actin in cortical lysates from PND1 mice of indicated genotypes relative to the normalized levels of each protein in control brains. Data show mean ± SEM for three biological replicates per genotype for one experiment. Unpaired t test. *p < 0.05. ( C ) Western blot analysis of total and surface levels of indicated proteins in DIV3 control and AnkB440 KO cortical neurons. ( D ) Quantification of total and surface protein levels normalized to actin relative to the normalized levels of each protein in control cortical neurons. Data show mean ± SEM for three biological replicates per genotype for one experiment. Unpaired t test. **p < 0.01. ( E ) Images show PLA signal between L1CAM and Nrp1 at the cell surface of the axon and GCs of DIV3 cortical neurons from the indicated genotypes. This assay used an Nrp1 antibody that selectively recognizes an extracellular epitope. Phalloidin staining was used to identify GCs. Scale bar, 2 μm. ( F ) Quantification of PLA signal at the GC surface relative to GC area collected from an average n = 15 GCs/genotype. The box and whisker plots represent all data points arranged from minimum to maximum. One-way ANOVA with Tukey’s post hoc analysis test for multiple comparisons. ****p < 0.0001. ( G ) Images show Nrp1 localization at the surface of GCs of DIV3 neurons untreated and treated with Sema 3 A, which induces the internalization of surface Nrp1. Scale bar, 1 μm. ( H ) Quantification of surface Nrp1 levels at GCs relative to GC area at the basal state and upon Sema 3A-induced Nrp1 internalization. Data represent mean ± SEM collected from an average of n = 90 GCs/treatment/genotype/experiment. Each dot represents one out of three independent experiments. One-way ANOVA with Tukey’s post hoc analysis test for multiple comparisons. **p < 0.01. Figure 7—source data 1. AnkB440 stabilizes the L1CAM-Nrp1 complex at the cell surface of GCs.

Article Snippet: Antibody , anti-Plexin A1 extracellular (Rabbit polyclonal) , Alomone , Cat# APR-081, RRID: AB_2756765 , WB (1:200).

Techniques: Western Blot, Expressing, Staining, Whisker Assay

Journal: eLife

Article Title: Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A

doi: 10.7554/eLife.69815

Figure Lengend Snippet:

Article Snippet: Antibody , anti-Plexin A1 extracellular (Rabbit polyclonal) , Alomone , Cat# APR-081, RRID: AB_2756765 , WB (1:200).

Techniques: Knock-Out, Recombinant, Mutagenesis, In Situ