ncam 1  (Alomone Labs)


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    Alomone Labs ncam 1
    NCAM -1 and ALCAM are enriched in the murine VCS. (A) Scatter plots identified <t>Ncam-1</t> and Alcam (red) as two of the most enriched genes with cell adhesion properties in mature PCs. (B) Quantitative RT-PCR (qPCR) confirmed significant Ncam-1 and Alcam enrichment in adult PCs in addition to other known markers of the VCS ( Cx40 and Cntn2 ) ( n =3). Error bars represent s.e.m. (C) Immunofluorescence staining in WT P21 murine hearts sections in the VCS shows overlapping expression of NCAM-1 (red) and ALCAM (red) with Cx40 (green) in the HPS. Scale bars: 25 μm.
    Ncam 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ncam 1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ncam 1 - by Bioz Stars, 2023-02
    93/100 stars

    Images

    1) Product Images from "Neural cell adhesion molecule is required for ventricular conduction system development"

    Article Title: Neural cell adhesion molecule is required for ventricular conduction system development

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.199431

    NCAM -1 and ALCAM are enriched in the murine VCS. (A) Scatter plots identified Ncam-1 and Alcam (red) as two of the most enriched genes with cell adhesion properties in mature PCs. (B) Quantitative RT-PCR (qPCR) confirmed significant Ncam-1 and Alcam enrichment in adult PCs in addition to other known markers of the VCS ( Cx40 and Cntn2 ) ( n =3). Error bars represent s.e.m. (C) Immunofluorescence staining in WT P21 murine hearts sections in the VCS shows overlapping expression of NCAM-1 (red) and ALCAM (red) with Cx40 (green) in the HPS. Scale bars: 25 μm.
    Figure Legend Snippet: NCAM -1 and ALCAM are enriched in the murine VCS. (A) Scatter plots identified Ncam-1 and Alcam (red) as two of the most enriched genes with cell adhesion properties in mature PCs. (B) Quantitative RT-PCR (qPCR) confirmed significant Ncam-1 and Alcam enrichment in adult PCs in addition to other known markers of the VCS ( Cx40 and Cntn2 ) ( n =3). Error bars represent s.e.m. (C) Immunofluorescence staining in WT P21 murine hearts sections in the VCS shows overlapping expression of NCAM-1 (red) and ALCAM (red) with Cx40 (green) in the HPS. Scale bars: 25 μm.

    Techniques Used: Quantitative RT-PCR, Immunofluorescence, Staining, Expressing

    Spatiotemporal expression of NCAM-1 and ALCAM during myocardial development. (A) Immunofluorescence staining of NCAM-1 (red) and Cx40 (green) during ventricular murine development at E10.5, E13.5 and E16.5. Right-hand panels show high-magnification views of the boxed areas. NCAM-1 is first detected in the compact myocardium at E13.5. By E16.5, NCAM-1 is expressed in the compact myocardium and a subset of Cx40 + cells in the trabecular myocardium. (B) Immunofluorescence staining for ALCAM (red) and Cx40 (green) during ventricular murine development at E10.5, E13.5 and E16.5. Right-hand panels show high-magnification views of the boxed areas. ALCAM is detected as early as E10.5 and its expression remains restricted to the trabecular myocardium. Scale bars: 100 μm (main panels); 25 μm (right-hand panels). (C) Immunofluorescence staining at P1 for NCAM-1 (green) and ALCAM (red) detected expression in trabecular ventricular myocardium, which becomes progressively restricted to the subendocardial region. By P7, NCAM-1 and ALCAM are restricted to PCs and absent from VMs. LV, left ventricle; RV, right ventricle. Scale bars: 25 μm.
    Figure Legend Snippet: Spatiotemporal expression of NCAM-1 and ALCAM during myocardial development. (A) Immunofluorescence staining of NCAM-1 (red) and Cx40 (green) during ventricular murine development at E10.5, E13.5 and E16.5. Right-hand panels show high-magnification views of the boxed areas. NCAM-1 is first detected in the compact myocardium at E13.5. By E16.5, NCAM-1 is expressed in the compact myocardium and a subset of Cx40 + cells in the trabecular myocardium. (B) Immunofluorescence staining for ALCAM (red) and Cx40 (green) during ventricular murine development at E10.5, E13.5 and E16.5. Right-hand panels show high-magnification views of the boxed areas. ALCAM is detected as early as E10.5 and its expression remains restricted to the trabecular myocardium. Scale bars: 100 μm (main panels); 25 μm (right-hand panels). (C) Immunofluorescence staining at P1 for NCAM-1 (green) and ALCAM (red) detected expression in trabecular ventricular myocardium, which becomes progressively restricted to the subendocardial region. By P7, NCAM-1 and ALCAM are restricted to PCs and absent from VMs. LV, left ventricle; RV, right ventricle. Scale bars: 25 μm.

    Techniques Used: Expressing, Immunofluorescence, Staining

    Ncam-1 -deficient mice display ventricular conduction slowing and a dysmorphic VCS morphology. (A) Representative surface ECG traces of adult Cntn2 , Alcam and Ncam-1 WT and KO mice. Ncam-1 KO mice show a modest but significant prolongation of the QRS interval compared with Ncam-1 WT controls. No difference in QRS was observed with Alcam - or Cntn2 -deficient mice. ( n =12 for each genotype). (B) Adult Cntn2 , Alcam and Ncam-1 KO mice backcrossed into the Cntn2 -EGFP reporter line were used to visualize the right and left HPS. Center panels are high-magnification views of the boxed areas of the left ventricle (LV). Right ventricle (RV) is displayed in the right-hand panels ( Cntn2 and Alcam KO n =5; Ncam-1 KO n =9, of which six had overt morphological defects). (C) RFP + cells dyed with TMRM were purified into ventricular (VM) and Purkinje (PC) fractions. Shown are representative FACS plots of PC (RFP + GFP + ) and VM (RFP + GFP − ) populations and the percentages of PCs relative to VMs were calculated for Ncam-1 WT and KO samples ( n =4). (D) PC population was also quantified using MATLAB in acquired GFP images from WT and Ncam-1 KO hearts. Pixel density was calculated per unit of masked region. All comparisons between WT and KO groups were made by applying a two-tailed Student's t -test ( n =4). Data represent mean±s.e.m. * P <0.05, *** P <0.001, WT versus KO. Scale bars: 1 mm.
    Figure Legend Snippet: Ncam-1 -deficient mice display ventricular conduction slowing and a dysmorphic VCS morphology. (A) Representative surface ECG traces of adult Cntn2 , Alcam and Ncam-1 WT and KO mice. Ncam-1 KO mice show a modest but significant prolongation of the QRS interval compared with Ncam-1 WT controls. No difference in QRS was observed with Alcam - or Cntn2 -deficient mice. ( n =12 for each genotype). (B) Adult Cntn2 , Alcam and Ncam-1 KO mice backcrossed into the Cntn2 -EGFP reporter line were used to visualize the right and left HPS. Center panels are high-magnification views of the boxed areas of the left ventricle (LV). Right ventricle (RV) is displayed in the right-hand panels ( Cntn2 and Alcam KO n =5; Ncam-1 KO n =9, of which six had overt morphological defects). (C) RFP + cells dyed with TMRM were purified into ventricular (VM) and Purkinje (PC) fractions. Shown are representative FACS plots of PC (RFP + GFP + ) and VM (RFP + GFP − ) populations and the percentages of PCs relative to VMs were calculated for Ncam-1 WT and KO samples ( n =4). (D) PC population was also quantified using MATLAB in acquired GFP images from WT and Ncam-1 KO hearts. Pixel density was calculated per unit of masked region. All comparisons between WT and KO groups were made by applying a two-tailed Student's t -test ( n =4). Data represent mean±s.e.m. * P <0.05, *** P <0.001, WT versus KO. Scale bars: 1 mm.

    Techniques Used: Purification, Two Tailed Test

    Dysregulation of PC programming in Ncam-1 KO mice. (A) Volcano plot of relative transcript expression from Ncam-1 WT and KO PCs ( n =3). All significantly ( P <0.05) expressed genes are labeled red (enriched) or blue (downregulated) and all nonsignificantly different transcripts are gray. (B) Functional network analysis using the DAVID functional annotation tool of differentially expressed genes from PCs of Ncam-1 WT and KO mice. (C) Table of selected targets and quantitative RT-PCR of enriched in PCs of Ncam-1 WT mice compared with PCs of Ncam-1 KO mice with log2-fold change, fold change and P -value. (D) SCN4B in PCs and VMs of Ncam-1 WT and KO mice ( n =3). Western blot analysis and quantification of adult brain (30 μg) and heart ventricular lysates (30 μg) probed for SCN4B. Vinculin was used as loading control. Data represent mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001 (two-tailed Student's t -test) WT versus KO. NS, not significant.
    Figure Legend Snippet: Dysregulation of PC programming in Ncam-1 KO mice. (A) Volcano plot of relative transcript expression from Ncam-1 WT and KO PCs ( n =3). All significantly ( P <0.05) expressed genes are labeled red (enriched) or blue (downregulated) and all nonsignificantly different transcripts are gray. (B) Functional network analysis using the DAVID functional annotation tool of differentially expressed genes from PCs of Ncam-1 WT and KO mice. (C) Table of selected targets and quantitative RT-PCR of enriched in PCs of Ncam-1 WT mice compared with PCs of Ncam-1 KO mice with log2-fold change, fold change and P -value. (D) SCN4B in PCs and VMs of Ncam-1 WT and KO mice ( n =3). Western blot analysis and quantification of adult brain (30 μg) and heart ventricular lysates (30 μg) probed for SCN4B. Vinculin was used as loading control. Data represent mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001 (two-tailed Student's t -test) WT versus KO. NS, not significant.

    Techniques Used: Expressing, Labeling, Functional Assay, Quantitative RT-PCR, Western Blot, Two Tailed Test

    PSA is expressed in the developing ventricular myocardium. (A) Western blot analysis of protein extract from whole ventricles at the specified developmental stages using anti-NCAM-1 and anti-PSA-NCAM-1 antibodies. Vinculin was used as loading control. Lysate protein amount loaded: Heart-PSA blot, 15 μg; Heart-NCAM-1 blot, 20 μg. (B) Immunofluorescence staining of NCAM-1 (red) and PSA (green) during ventricular murine development at E13.5, E14.5 and E16.5. PSA is first detected in the atria, AV junction and epicardium at E13.5. By E14.5 and for the remainder of embryonic development, PSA is also detected in NCAM-1 + trabecular cardiomyocytes in the subendocardial region. On the right, high-magnification images of the boxed area at E16.5 shows that PSA (green) and NCAM-1 (red) colocalize in the cell membrane. (C) Immunofluorescence staining of E16.5 Ncam-1 KO sequential sections shows no PSA expression in the absence of NCAM-1. (D) Tyrosine hydroxylase expression in AV junction at P3. Immunofluorescence staining of NCAM-1 (red) with TH (green) and PSA (green) at P3 showing atrioventricular junction region. Right-hand panels show high-magnification views of the boxed areas. AV, atrioventricular junction; LV, left ventricle; RV, right ventricle. Scale bars: 100 μm (B, main panels; C; D, left); 25 μm (B, high-magnification panel; D, right).
    Figure Legend Snippet: PSA is expressed in the developing ventricular myocardium. (A) Western blot analysis of protein extract from whole ventricles at the specified developmental stages using anti-NCAM-1 and anti-PSA-NCAM-1 antibodies. Vinculin was used as loading control. Lysate protein amount loaded: Heart-PSA blot, 15 μg; Heart-NCAM-1 blot, 20 μg. (B) Immunofluorescence staining of NCAM-1 (red) and PSA (green) during ventricular murine development at E13.5, E14.5 and E16.5. PSA is first detected in the atria, AV junction and epicardium at E13.5. By E14.5 and for the remainder of embryonic development, PSA is also detected in NCAM-1 + trabecular cardiomyocytes in the subendocardial region. On the right, high-magnification images of the boxed area at E16.5 shows that PSA (green) and NCAM-1 (red) colocalize in the cell membrane. (C) Immunofluorescence staining of E16.5 Ncam-1 KO sequential sections shows no PSA expression in the absence of NCAM-1. (D) Tyrosine hydroxylase expression in AV junction at P3. Immunofluorescence staining of NCAM-1 (red) with TH (green) and PSA (green) at P3 showing atrioventricular junction region. Right-hand panels show high-magnification views of the boxed areas. AV, atrioventricular junction; LV, left ventricle; RV, right ventricle. Scale bars: 100 μm (B, main panels; C; D, left); 25 μm (B, high-magnification panel; D, right).

    Techniques Used: Western Blot, Immunofluorescence, Staining, Expressing

    Generation of PSA-deficient double KO mice by CRISPR/Cas9. (A) ST8Sia2 and ST8Sia4 sgRNA (blue) targeting sequences. Stop codon is labeled with a star and protospacer adjacent motif (PAM) sequence is indicated in red. Black arrowheads indicate the site of the mutation, which is indicated in parentheses for each. Base pair sequence indicates the region where the mutation is, which is indicated with an arrow in the KO sequences by Sanger sequencing. (B) Western blot of protein extract from whole ventricles from PSA-deficient DKOs and littermate controls at P1 (10 μg). Results show that PSA is not detected in DKO mice but is still detected in Sia2 +/− /Sia4 +/− , Sia2 −/− /Sia4 +/− and Sia2 +/− /Sia4 −/− littermates. Probing with anti-NCAM-1 antibody reveals unaffected NCAM-1 levels for all heart samples. Vinculin was used as loading control. (C) Body weight and heart weight/tibia length (HR/TL) ratios of Ctrl (DHet) and PSA-deficient DKO hearts. Hematoxylin and Eosin and trichrome stain of Ctrl (DHet) and PSA-deficient DKO hearts is shown below. Data represent mean±s.e.m. *** P <0.001, **** P <0.0001 (two-tailed Student's t -test) of Ctrl (DHet) versus DKO. Scale bar: 1 mm.
    Figure Legend Snippet: Generation of PSA-deficient double KO mice by CRISPR/Cas9. (A) ST8Sia2 and ST8Sia4 sgRNA (blue) targeting sequences. Stop codon is labeled with a star and protospacer adjacent motif (PAM) sequence is indicated in red. Black arrowheads indicate the site of the mutation, which is indicated in parentheses for each. Base pair sequence indicates the region where the mutation is, which is indicated with an arrow in the KO sequences by Sanger sequencing. (B) Western blot of protein extract from whole ventricles from PSA-deficient DKOs and littermate controls at P1 (10 μg). Results show that PSA is not detected in DKO mice but is still detected in Sia2 +/− /Sia4 +/− , Sia2 −/− /Sia4 +/− and Sia2 +/− /Sia4 −/− littermates. Probing with anti-NCAM-1 antibody reveals unaffected NCAM-1 levels for all heart samples. Vinculin was used as loading control. (C) Body weight and heart weight/tibia length (HR/TL) ratios of Ctrl (DHet) and PSA-deficient DKO hearts. Hematoxylin and Eosin and trichrome stain of Ctrl (DHet) and PSA-deficient DKO hearts is shown below. Data represent mean±s.e.m. *** P <0.001, **** P <0.0001 (two-tailed Student's t -test) of Ctrl (DHet) versus DKO. Scale bar: 1 mm.

    Techniques Used: CRISPR, Labeling, Sequencing, Mutagenesis, Western Blot, Staining, Two Tailed Test

    Mislocalization of cell membrane proteins of PCs in PSA-deficient mice. (A) Immunofluorescence staining of P21 WT and PSA-deficient hearts sections with antibodies against NCAM-1 (red) and CNTN2 (green). In WT sections, PCs show colocalization of CNTN2 and NCAM-1 in the intercalated disc. PSA-deficient mice show nonoverlapping regions of CNTN2 and NCAM-1. Insets are magnified images of the dashed boxed regions. (B) Immunofluorescence staining of PCs of WT and PSA-deficient P21 hearts with antibodies against Cx40 (red) and ALCAM (green). Insets are magnified images of the dashed boxed regions showing ALCAM + PCs that are negative for Cx40. (C) Immunofluorescence staining shows incomplete localization of Cx40 or NCAM-1 (red) to the cell membrane delineated by WGA (green) in DKO PSA-deficient murine hearts at P21. Insets are magnified images of dashed boxed regions. (D) ECGs of PSA-deficient DKO ( n =10) mice and littermates with allelic combinations: DHet ( n =10), Het/KO ( n =10) and KO/Het ( n =8). PSA-deficient DKO mice displayed prolonged QRS duration compared with all three other allelic variants. Data represent mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.0001 (one-way ANOVA). (E) Left and right ventricle whole-mount dissections of DHet and PSA-deficient DKO mice in the Cntn2-EGFP background. LV, left ventricle; RV, right ventricle. (F) Representative images of 3D SEM sections of DKO and DHet P21 heart sections. Yellow arrows point to cell junctions. Color scale indicates the thickness of the three-dimensional mesh. The colors ranging from purple to yellow represent narrower to wider volumes between membranes, respectively. Scale bars: 25 μm (A-C); 1 mm (E); 1 μm (F).
    Figure Legend Snippet: Mislocalization of cell membrane proteins of PCs in PSA-deficient mice. (A) Immunofluorescence staining of P21 WT and PSA-deficient hearts sections with antibodies against NCAM-1 (red) and CNTN2 (green). In WT sections, PCs show colocalization of CNTN2 and NCAM-1 in the intercalated disc. PSA-deficient mice show nonoverlapping regions of CNTN2 and NCAM-1. Insets are magnified images of the dashed boxed regions. (B) Immunofluorescence staining of PCs of WT and PSA-deficient P21 hearts with antibodies against Cx40 (red) and ALCAM (green). Insets are magnified images of the dashed boxed regions showing ALCAM + PCs that are negative for Cx40. (C) Immunofluorescence staining shows incomplete localization of Cx40 or NCAM-1 (red) to the cell membrane delineated by WGA (green) in DKO PSA-deficient murine hearts at P21. Insets are magnified images of dashed boxed regions. (D) ECGs of PSA-deficient DKO ( n =10) mice and littermates with allelic combinations: DHet ( n =10), Het/KO ( n =10) and KO/Het ( n =8). PSA-deficient DKO mice displayed prolonged QRS duration compared with all three other allelic variants. Data represent mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.0001 (one-way ANOVA). (E) Left and right ventricle whole-mount dissections of DHet and PSA-deficient DKO mice in the Cntn2-EGFP background. LV, left ventricle; RV, right ventricle. (F) Representative images of 3D SEM sections of DKO and DHet P21 heart sections. Yellow arrows point to cell junctions. Color scale indicates the thickness of the three-dimensional mesh. The colors ranging from purple to yellow represent narrower to wider volumes between membranes, respectively. Scale bars: 25 μm (A-C); 1 mm (E); 1 μm (F).

    Techniques Used: Immunofluorescence, Staining

    ncam 1  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
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  • 93

    Structured Review

    Alomone Labs ncam 1
    NCAM -1 and ALCAM are enriched in the murine VCS. (A) Scatter plots identified <t>Ncam-1</t> and Alcam (red) as two of the most enriched genes with cell adhesion properties in mature PCs. (B) Quantitative RT-PCR (qPCR) confirmed significant Ncam-1 and Alcam enrichment in adult PCs in addition to other known markers of the VCS ( Cx40 and Cntn2 ) ( n =3). Error bars represent s.e.m. (C) Immunofluorescence staining in WT P21 murine hearts sections in the VCS shows overlapping expression of NCAM-1 (red) and ALCAM (red) with Cx40 (green) in the HPS. Scale bars: 25 μm.
    Ncam 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ncam 1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ncam 1 - by Bioz Stars, 2023-02
    93/100 stars

    Images

    1) Product Images from "Neural cell adhesion molecule is required for ventricular conduction system development"

    Article Title: Neural cell adhesion molecule is required for ventricular conduction system development

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.199431

    NCAM -1 and ALCAM are enriched in the murine VCS. (A) Scatter plots identified Ncam-1 and Alcam (red) as two of the most enriched genes with cell adhesion properties in mature PCs. (B) Quantitative RT-PCR (qPCR) confirmed significant Ncam-1 and Alcam enrichment in adult PCs in addition to other known markers of the VCS ( Cx40 and Cntn2 ) ( n =3). Error bars represent s.e.m. (C) Immunofluorescence staining in WT P21 murine hearts sections in the VCS shows overlapping expression of NCAM-1 (red) and ALCAM (red) with Cx40 (green) in the HPS. Scale bars: 25 μm.
    Figure Legend Snippet: NCAM -1 and ALCAM are enriched in the murine VCS. (A) Scatter plots identified Ncam-1 and Alcam (red) as two of the most enriched genes with cell adhesion properties in mature PCs. (B) Quantitative RT-PCR (qPCR) confirmed significant Ncam-1 and Alcam enrichment in adult PCs in addition to other known markers of the VCS ( Cx40 and Cntn2 ) ( n =3). Error bars represent s.e.m. (C) Immunofluorescence staining in WT P21 murine hearts sections in the VCS shows overlapping expression of NCAM-1 (red) and ALCAM (red) with Cx40 (green) in the HPS. Scale bars: 25 μm.

    Techniques Used: Quantitative RT-PCR, Immunofluorescence, Staining, Expressing

    Spatiotemporal expression of NCAM-1 and ALCAM during myocardial development. (A) Immunofluorescence staining of NCAM-1 (red) and Cx40 (green) during ventricular murine development at E10.5, E13.5 and E16.5. Right-hand panels show high-magnification views of the boxed areas. NCAM-1 is first detected in the compact myocardium at E13.5. By E16.5, NCAM-1 is expressed in the compact myocardium and a subset of Cx40 + cells in the trabecular myocardium. (B) Immunofluorescence staining for ALCAM (red) and Cx40 (green) during ventricular murine development at E10.5, E13.5 and E16.5. Right-hand panels show high-magnification views of the boxed areas. ALCAM is detected as early as E10.5 and its expression remains restricted to the trabecular myocardium. Scale bars: 100 μm (main panels); 25 μm (right-hand panels). (C) Immunofluorescence staining at P1 for NCAM-1 (green) and ALCAM (red) detected expression in trabecular ventricular myocardium, which becomes progressively restricted to the subendocardial region. By P7, NCAM-1 and ALCAM are restricted to PCs and absent from VMs. LV, left ventricle; RV, right ventricle. Scale bars: 25 μm.
    Figure Legend Snippet: Spatiotemporal expression of NCAM-1 and ALCAM during myocardial development. (A) Immunofluorescence staining of NCAM-1 (red) and Cx40 (green) during ventricular murine development at E10.5, E13.5 and E16.5. Right-hand panels show high-magnification views of the boxed areas. NCAM-1 is first detected in the compact myocardium at E13.5. By E16.5, NCAM-1 is expressed in the compact myocardium and a subset of Cx40 + cells in the trabecular myocardium. (B) Immunofluorescence staining for ALCAM (red) and Cx40 (green) during ventricular murine development at E10.5, E13.5 and E16.5. Right-hand panels show high-magnification views of the boxed areas. ALCAM is detected as early as E10.5 and its expression remains restricted to the trabecular myocardium. Scale bars: 100 μm (main panels); 25 μm (right-hand panels). (C) Immunofluorescence staining at P1 for NCAM-1 (green) and ALCAM (red) detected expression in trabecular ventricular myocardium, which becomes progressively restricted to the subendocardial region. By P7, NCAM-1 and ALCAM are restricted to PCs and absent from VMs. LV, left ventricle; RV, right ventricle. Scale bars: 25 μm.

    Techniques Used: Expressing, Immunofluorescence, Staining

    Ncam-1 -deficient mice display ventricular conduction slowing and a dysmorphic VCS morphology. (A) Representative surface ECG traces of adult Cntn2 , Alcam and Ncam-1 WT and KO mice. Ncam-1 KO mice show a modest but significant prolongation of the QRS interval compared with Ncam-1 WT controls. No difference in QRS was observed with Alcam - or Cntn2 -deficient mice. ( n =12 for each genotype). (B) Adult Cntn2 , Alcam and Ncam-1 KO mice backcrossed into the Cntn2 -EGFP reporter line were used to visualize the right and left HPS. Center panels are high-magnification views of the boxed areas of the left ventricle (LV). Right ventricle (RV) is displayed in the right-hand panels ( Cntn2 and Alcam KO n =5; Ncam-1 KO n =9, of which six had overt morphological defects). (C) RFP + cells dyed with TMRM were purified into ventricular (VM) and Purkinje (PC) fractions. Shown are representative FACS plots of PC (RFP + GFP + ) and VM (RFP + GFP − ) populations and the percentages of PCs relative to VMs were calculated for Ncam-1 WT and KO samples ( n =4). (D) PC population was also quantified using MATLAB in acquired GFP images from WT and Ncam-1 KO hearts. Pixel density was calculated per unit of masked region. All comparisons between WT and KO groups were made by applying a two-tailed Student's t -test ( n =4). Data represent mean±s.e.m. * P <0.05, *** P <0.001, WT versus KO. Scale bars: 1 mm.
    Figure Legend Snippet: Ncam-1 -deficient mice display ventricular conduction slowing and a dysmorphic VCS morphology. (A) Representative surface ECG traces of adult Cntn2 , Alcam and Ncam-1 WT and KO mice. Ncam-1 KO mice show a modest but significant prolongation of the QRS interval compared with Ncam-1 WT controls. No difference in QRS was observed with Alcam - or Cntn2 -deficient mice. ( n =12 for each genotype). (B) Adult Cntn2 , Alcam and Ncam-1 KO mice backcrossed into the Cntn2 -EGFP reporter line were used to visualize the right and left HPS. Center panels are high-magnification views of the boxed areas of the left ventricle (LV). Right ventricle (RV) is displayed in the right-hand panels ( Cntn2 and Alcam KO n =5; Ncam-1 KO n =9, of which six had overt morphological defects). (C) RFP + cells dyed with TMRM were purified into ventricular (VM) and Purkinje (PC) fractions. Shown are representative FACS plots of PC (RFP + GFP + ) and VM (RFP + GFP − ) populations and the percentages of PCs relative to VMs were calculated for Ncam-1 WT and KO samples ( n =4). (D) PC population was also quantified using MATLAB in acquired GFP images from WT and Ncam-1 KO hearts. Pixel density was calculated per unit of masked region. All comparisons between WT and KO groups were made by applying a two-tailed Student's t -test ( n =4). Data represent mean±s.e.m. * P <0.05, *** P <0.001, WT versus KO. Scale bars: 1 mm.

    Techniques Used: Purification, Two Tailed Test

    Dysregulation of PC programming in Ncam-1 KO mice. (A) Volcano plot of relative transcript expression from Ncam-1 WT and KO PCs ( n =3). All significantly ( P <0.05) expressed genes are labeled red (enriched) or blue (downregulated) and all nonsignificantly different transcripts are gray. (B) Functional network analysis using the DAVID functional annotation tool of differentially expressed genes from PCs of Ncam-1 WT and KO mice. (C) Table of selected targets and quantitative RT-PCR of enriched in PCs of Ncam-1 WT mice compared with PCs of Ncam-1 KO mice with log2-fold change, fold change and P -value. (D) SCN4B in PCs and VMs of Ncam-1 WT and KO mice ( n =3). Western blot analysis and quantification of adult brain (30 μg) and heart ventricular lysates (30 μg) probed for SCN4B. Vinculin was used as loading control. Data represent mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001 (two-tailed Student's t -test) WT versus KO. NS, not significant.
    Figure Legend Snippet: Dysregulation of PC programming in Ncam-1 KO mice. (A) Volcano plot of relative transcript expression from Ncam-1 WT and KO PCs ( n =3). All significantly ( P <0.05) expressed genes are labeled red (enriched) or blue (downregulated) and all nonsignificantly different transcripts are gray. (B) Functional network analysis using the DAVID functional annotation tool of differentially expressed genes from PCs of Ncam-1 WT and KO mice. (C) Table of selected targets and quantitative RT-PCR of enriched in PCs of Ncam-1 WT mice compared with PCs of Ncam-1 KO mice with log2-fold change, fold change and P -value. (D) SCN4B in PCs and VMs of Ncam-1 WT and KO mice ( n =3). Western blot analysis and quantification of adult brain (30 μg) and heart ventricular lysates (30 μg) probed for SCN4B. Vinculin was used as loading control. Data represent mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001 (two-tailed Student's t -test) WT versus KO. NS, not significant.

    Techniques Used: Expressing, Labeling, Functional Assay, Quantitative RT-PCR, Western Blot, Two Tailed Test

    PSA is expressed in the developing ventricular myocardium. (A) Western blot analysis of protein extract from whole ventricles at the specified developmental stages using anti-NCAM-1 and anti-PSA-NCAM-1 antibodies. Vinculin was used as loading control. Lysate protein amount loaded: Heart-PSA blot, 15 μg; Heart-NCAM-1 blot, 20 μg. (B) Immunofluorescence staining of NCAM-1 (red) and PSA (green) during ventricular murine development at E13.5, E14.5 and E16.5. PSA is first detected in the atria, AV junction and epicardium at E13.5. By E14.5 and for the remainder of embryonic development, PSA is also detected in NCAM-1 + trabecular cardiomyocytes in the subendocardial region. On the right, high-magnification images of the boxed area at E16.5 shows that PSA (green) and NCAM-1 (red) colocalize in the cell membrane. (C) Immunofluorescence staining of E16.5 Ncam-1 KO sequential sections shows no PSA expression in the absence of NCAM-1. (D) Tyrosine hydroxylase expression in AV junction at P3. Immunofluorescence staining of NCAM-1 (red) with TH (green) and PSA (green) at P3 showing atrioventricular junction region. Right-hand panels show high-magnification views of the boxed areas. AV, atrioventricular junction; LV, left ventricle; RV, right ventricle. Scale bars: 100 μm (B, main panels; C; D, left); 25 μm (B, high-magnification panel; D, right).
    Figure Legend Snippet: PSA is expressed in the developing ventricular myocardium. (A) Western blot analysis of protein extract from whole ventricles at the specified developmental stages using anti-NCAM-1 and anti-PSA-NCAM-1 antibodies. Vinculin was used as loading control. Lysate protein amount loaded: Heart-PSA blot, 15 μg; Heart-NCAM-1 blot, 20 μg. (B) Immunofluorescence staining of NCAM-1 (red) and PSA (green) during ventricular murine development at E13.5, E14.5 and E16.5. PSA is first detected in the atria, AV junction and epicardium at E13.5. By E14.5 and for the remainder of embryonic development, PSA is also detected in NCAM-1 + trabecular cardiomyocytes in the subendocardial region. On the right, high-magnification images of the boxed area at E16.5 shows that PSA (green) and NCAM-1 (red) colocalize in the cell membrane. (C) Immunofluorescence staining of E16.5 Ncam-1 KO sequential sections shows no PSA expression in the absence of NCAM-1. (D) Tyrosine hydroxylase expression in AV junction at P3. Immunofluorescence staining of NCAM-1 (red) with TH (green) and PSA (green) at P3 showing atrioventricular junction region. Right-hand panels show high-magnification views of the boxed areas. AV, atrioventricular junction; LV, left ventricle; RV, right ventricle. Scale bars: 100 μm (B, main panels; C; D, left); 25 μm (B, high-magnification panel; D, right).

    Techniques Used: Western Blot, Immunofluorescence, Staining, Expressing

    Generation of PSA-deficient double KO mice by CRISPR/Cas9. (A) ST8Sia2 and ST8Sia4 sgRNA (blue) targeting sequences. Stop codon is labeled with a star and protospacer adjacent motif (PAM) sequence is indicated in red. Black arrowheads indicate the site of the mutation, which is indicated in parentheses for each. Base pair sequence indicates the region where the mutation is, which is indicated with an arrow in the KO sequences by Sanger sequencing. (B) Western blot of protein extract from whole ventricles from PSA-deficient DKOs and littermate controls at P1 (10 μg). Results show that PSA is not detected in DKO mice but is still detected in Sia2 +/− /Sia4 +/− , Sia2 −/− /Sia4 +/− and Sia2 +/− /Sia4 −/− littermates. Probing with anti-NCAM-1 antibody reveals unaffected NCAM-1 levels for all heart samples. Vinculin was used as loading control. (C) Body weight and heart weight/tibia length (HR/TL) ratios of Ctrl (DHet) and PSA-deficient DKO hearts. Hematoxylin and Eosin and trichrome stain of Ctrl (DHet) and PSA-deficient DKO hearts is shown below. Data represent mean±s.e.m. *** P <0.001, **** P <0.0001 (two-tailed Student's t -test) of Ctrl (DHet) versus DKO. Scale bar: 1 mm.
    Figure Legend Snippet: Generation of PSA-deficient double KO mice by CRISPR/Cas9. (A) ST8Sia2 and ST8Sia4 sgRNA (blue) targeting sequences. Stop codon is labeled with a star and protospacer adjacent motif (PAM) sequence is indicated in red. Black arrowheads indicate the site of the mutation, which is indicated in parentheses for each. Base pair sequence indicates the region where the mutation is, which is indicated with an arrow in the KO sequences by Sanger sequencing. (B) Western blot of protein extract from whole ventricles from PSA-deficient DKOs and littermate controls at P1 (10 μg). Results show that PSA is not detected in DKO mice but is still detected in Sia2 +/− /Sia4 +/− , Sia2 −/− /Sia4 +/− and Sia2 +/− /Sia4 −/− littermates. Probing with anti-NCAM-1 antibody reveals unaffected NCAM-1 levels for all heart samples. Vinculin was used as loading control. (C) Body weight and heart weight/tibia length (HR/TL) ratios of Ctrl (DHet) and PSA-deficient DKO hearts. Hematoxylin and Eosin and trichrome stain of Ctrl (DHet) and PSA-deficient DKO hearts is shown below. Data represent mean±s.e.m. *** P <0.001, **** P <0.0001 (two-tailed Student's t -test) of Ctrl (DHet) versus DKO. Scale bar: 1 mm.

    Techniques Used: CRISPR, Labeling, Sequencing, Mutagenesis, Western Blot, Staining, Two Tailed Test

    Mislocalization of cell membrane proteins of PCs in PSA-deficient mice. (A) Immunofluorescence staining of P21 WT and PSA-deficient hearts sections with antibodies against NCAM-1 (red) and CNTN2 (green). In WT sections, PCs show colocalization of CNTN2 and NCAM-1 in the intercalated disc. PSA-deficient mice show nonoverlapping regions of CNTN2 and NCAM-1. Insets are magnified images of the dashed boxed regions. (B) Immunofluorescence staining of PCs of WT and PSA-deficient P21 hearts with antibodies against Cx40 (red) and ALCAM (green). Insets are magnified images of the dashed boxed regions showing ALCAM + PCs that are negative for Cx40. (C) Immunofluorescence staining shows incomplete localization of Cx40 or NCAM-1 (red) to the cell membrane delineated by WGA (green) in DKO PSA-deficient murine hearts at P21. Insets are magnified images of dashed boxed regions. (D) ECGs of PSA-deficient DKO ( n =10) mice and littermates with allelic combinations: DHet ( n =10), Het/KO ( n =10) and KO/Het ( n =8). PSA-deficient DKO mice displayed prolonged QRS duration compared with all three other allelic variants. Data represent mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.0001 (one-way ANOVA). (E) Left and right ventricle whole-mount dissections of DHet and PSA-deficient DKO mice in the Cntn2-EGFP background. LV, left ventricle; RV, right ventricle. (F) Representative images of 3D SEM sections of DKO and DHet P21 heart sections. Yellow arrows point to cell junctions. Color scale indicates the thickness of the three-dimensional mesh. The colors ranging from purple to yellow represent narrower to wider volumes between membranes, respectively. Scale bars: 25 μm (A-C); 1 mm (E); 1 μm (F).
    Figure Legend Snippet: Mislocalization of cell membrane proteins of PCs in PSA-deficient mice. (A) Immunofluorescence staining of P21 WT and PSA-deficient hearts sections with antibodies against NCAM-1 (red) and CNTN2 (green). In WT sections, PCs show colocalization of CNTN2 and NCAM-1 in the intercalated disc. PSA-deficient mice show nonoverlapping regions of CNTN2 and NCAM-1. Insets are magnified images of the dashed boxed regions. (B) Immunofluorescence staining of PCs of WT and PSA-deficient P21 hearts with antibodies against Cx40 (red) and ALCAM (green). Insets are magnified images of the dashed boxed regions showing ALCAM + PCs that are negative for Cx40. (C) Immunofluorescence staining shows incomplete localization of Cx40 or NCAM-1 (red) to the cell membrane delineated by WGA (green) in DKO PSA-deficient murine hearts at P21. Insets are magnified images of dashed boxed regions. (D) ECGs of PSA-deficient DKO ( n =10) mice and littermates with allelic combinations: DHet ( n =10), Het/KO ( n =10) and KO/Het ( n =8). PSA-deficient DKO mice displayed prolonged QRS duration compared with all three other allelic variants. Data represent mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.0001 (one-way ANOVA). (E) Left and right ventricle whole-mount dissections of DHet and PSA-deficient DKO mice in the Cntn2-EGFP background. LV, left ventricle; RV, right ventricle. (F) Representative images of 3D SEM sections of DKO and DHet P21 heart sections. Yellow arrows point to cell junctions. Color scale indicates the thickness of the three-dimensional mesh. The colors ranging from purple to yellow represent narrower to wider volumes between membranes, respectively. Scale bars: 25 μm (A-C); 1 mm (E); 1 μm (F).

    Techniques Used: Immunofluorescence, Staining

    ncam  (Alomone Labs)


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    Alomone Labs ncam
    (A) RT-PCR <t>for</t> <t>trpm8</t> (236 bp) from cDNA extracted from stage 24 whole embryo or dissected spinal cord. +/− RT, in the presence or absence of the reverse transcriptase, respectively, during conversion of isolated mRNA into cDNA. (B) In situ hybridization for trpm8 in stage 24 embryos showing specific labeling in brain and spinal cord. (C) Western blot assays from egg, stage 24 wild-type whole embryo or from stage 40 whole larva, control morpholino (MO) or TRPM8-translation-blockingMO 1 (TRPM8-tbMO1) lysates. Predicted TRPM8 molecular weight [MW]: 132 kDa. Shown are representative examples of one of 3 independent experiments. β-tubulin was used as loading control. (D) Immunostained transverse section of stage 25 spinal cord (outlined). D, dorsal; V, ventral; scale bar, 20 μm; arrows indicate TRPM8 clusters in ventral neuron domains. <t>NCAM</t> labeling was used as counterstaining. (E) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged at 1 Hz for 90 s. Either 100 μM (−)-menthol or vehicle (0.05% DMSO)was added after 35 s of imaging and recording continued for another 60 s. Images show a menthol-responsive ventral neuron before (left, control) and after (right) addition of (−)-menthol. Colored scale shows fluorescence intensity in arbitrary units. Traces show the changes in fluorescence for the indicated cell (arrow) in both trials. (F) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged in 30-min intervals at cold (14.5°C) and warm (26.5°C) temperatures in the absence (vehicle, 0.1% DMSO) or presence of 10 μM AMTB, TRPM8 inhibitor. Scatterplots show changes in Ca 2+ spike frequency when switching temperatures in individual spinal neurons and geometric mean (black lines) from N = 3 ventral spinal cords per condition (n of neurons analyzed: DMSO, 62; AMTB, 71). Teal circles represent neurons with higher spike frequency at 14.5°C, magenta circles represent neurons with higher spike frequency at 26.5°C, and black circles represent neurons with no change in spike frequency across temperatures; ****p < 0.0001, comparison within treatments Wilcoxon matched-pairs signed rank, two-tailed test. (G) RT-PCR from cDNA collected from stage 46 larvae previously injected with 2.5 pmol standard control morpholino (Control-MO) or TRPM8-splicing-blocking morpholino (TRPM8-sbMO) shows that trpm8 mature transcript (349 bp) is not detected in TRPM8-sbMO animals. odc : ornithine decarboxylase (101 bp) as positive control. (H) TRPM8-sbMO or Control-MO containing spinal cord from stage 24 embryos were Ca 2+ -imaged for 30 min at cold temperature (14.5°C). Graph shows individual (scatterplots) and geometric mean (black lines) Ca 2+ spike frequency from N = 3 ventral spinal cords pergroup (n of neurons analyzed: Control-MO, 81; TRPM8-sbMO, 64), ****p < 0.001, Kolmogorov-Smirnov, two-tailed test. See also and .
    Ncam, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Growth at Cold Temperature Increases the Number of Motor Neurons to Optimize Locomotor Function"

    Article Title: Growth at Cold Temperature Increases the Number of Motor Neurons to Optimize Locomotor Function

    Journal: Current biology : CB

    doi: 10.1016/j.cub.2019.04.072

    (A) RT-PCR for trpm8 (236 bp) from cDNA extracted from stage 24 whole embryo or dissected spinal cord. +/− RT, in the presence or absence of the reverse transcriptase, respectively, during conversion of isolated mRNA into cDNA. (B) In situ hybridization for trpm8 in stage 24 embryos showing specific labeling in brain and spinal cord. (C) Western blot assays from egg, stage 24 wild-type whole embryo or from stage 40 whole larva, control morpholino (MO) or TRPM8-translation-blockingMO 1 (TRPM8-tbMO1) lysates. Predicted TRPM8 molecular weight [MW]: 132 kDa. Shown are representative examples of one of 3 independent experiments. β-tubulin was used as loading control. (D) Immunostained transverse section of stage 25 spinal cord (outlined). D, dorsal; V, ventral; scale bar, 20 μm; arrows indicate TRPM8 clusters in ventral neuron domains. NCAM labeling was used as counterstaining. (E) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged at 1 Hz for 90 s. Either 100 μM (−)-menthol or vehicle (0.05% DMSO)was added after 35 s of imaging and recording continued for another 60 s. Images show a menthol-responsive ventral neuron before (left, control) and after (right) addition of (−)-menthol. Colored scale shows fluorescence intensity in arbitrary units. Traces show the changes in fluorescence for the indicated cell (arrow) in both trials. (F) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged in 30-min intervals at cold (14.5°C) and warm (26.5°C) temperatures in the absence (vehicle, 0.1% DMSO) or presence of 10 μM AMTB, TRPM8 inhibitor. Scatterplots show changes in Ca 2+ spike frequency when switching temperatures in individual spinal neurons and geometric mean (black lines) from N = 3 ventral spinal cords per condition (n of neurons analyzed: DMSO, 62; AMTB, 71). Teal circles represent neurons with higher spike frequency at 14.5°C, magenta circles represent neurons with higher spike frequency at 26.5°C, and black circles represent neurons with no change in spike frequency across temperatures; ****p < 0.0001, comparison within treatments Wilcoxon matched-pairs signed rank, two-tailed test. (G) RT-PCR from cDNA collected from stage 46 larvae previously injected with 2.5 pmol standard control morpholino (Control-MO) or TRPM8-splicing-blocking morpholino (TRPM8-sbMO) shows that trpm8 mature transcript (349 bp) is not detected in TRPM8-sbMO animals. odc : ornithine decarboxylase (101 bp) as positive control. (H) TRPM8-sbMO or Control-MO containing spinal cord from stage 24 embryos were Ca 2+ -imaged for 30 min at cold temperature (14.5°C). Graph shows individual (scatterplots) and geometric mean (black lines) Ca 2+ spike frequency from N = 3 ventral spinal cords pergroup (n of neurons analyzed: Control-MO, 81; TRPM8-sbMO, 64), ****p < 0.001, Kolmogorov-Smirnov, two-tailed test. See also and .
    Figure Legend Snippet: (A) RT-PCR for trpm8 (236 bp) from cDNA extracted from stage 24 whole embryo or dissected spinal cord. +/− RT, in the presence or absence of the reverse transcriptase, respectively, during conversion of isolated mRNA into cDNA. (B) In situ hybridization for trpm8 in stage 24 embryos showing specific labeling in brain and spinal cord. (C) Western blot assays from egg, stage 24 wild-type whole embryo or from stage 40 whole larva, control morpholino (MO) or TRPM8-translation-blockingMO 1 (TRPM8-tbMO1) lysates. Predicted TRPM8 molecular weight [MW]: 132 kDa. Shown are representative examples of one of 3 independent experiments. β-tubulin was used as loading control. (D) Immunostained transverse section of stage 25 spinal cord (outlined). D, dorsal; V, ventral; scale bar, 20 μm; arrows indicate TRPM8 clusters in ventral neuron domains. NCAM labeling was used as counterstaining. (E) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged at 1 Hz for 90 s. Either 100 μM (−)-menthol or vehicle (0.05% DMSO)was added after 35 s of imaging and recording continued for another 60 s. Images show a menthol-responsive ventral neuron before (left, control) and after (right) addition of (−)-menthol. Colored scale shows fluorescence intensity in arbitrary units. Traces show the changes in fluorescence for the indicated cell (arrow) in both trials. (F) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged in 30-min intervals at cold (14.5°C) and warm (26.5°C) temperatures in the absence (vehicle, 0.1% DMSO) or presence of 10 μM AMTB, TRPM8 inhibitor. Scatterplots show changes in Ca 2+ spike frequency when switching temperatures in individual spinal neurons and geometric mean (black lines) from N = 3 ventral spinal cords per condition (n of neurons analyzed: DMSO, 62; AMTB, 71). Teal circles represent neurons with higher spike frequency at 14.5°C, magenta circles represent neurons with higher spike frequency at 26.5°C, and black circles represent neurons with no change in spike frequency across temperatures; ****p < 0.0001, comparison within treatments Wilcoxon matched-pairs signed rank, two-tailed test. (G) RT-PCR from cDNA collected from stage 46 larvae previously injected with 2.5 pmol standard control morpholino (Control-MO) or TRPM8-splicing-blocking morpholino (TRPM8-sbMO) shows that trpm8 mature transcript (349 bp) is not detected in TRPM8-sbMO animals. odc : ornithine decarboxylase (101 bp) as positive control. (H) TRPM8-sbMO or Control-MO containing spinal cord from stage 24 embryos were Ca 2+ -imaged for 30 min at cold temperature (14.5°C). Graph shows individual (scatterplots) and geometric mean (black lines) Ca 2+ spike frequency from N = 3 ventral spinal cords pergroup (n of neurons analyzed: Control-MO, 81; TRPM8-sbMO, 64), ****p < 0.001, Kolmogorov-Smirnov, two-tailed test. See also and .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, In Situ Hybridization, Labeling, Western Blot, Molecular Weight, Imaging, Fluorescence, Two Tailed Test, Injection, Blocking Assay, Positive Control

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Western Blot, In Situ, Luciferase, Reporter Assay, Isolation, Sequencing, Software

    anti psa ncam antibody  (Alomone Labs)


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    Alomone Labs anti psa ncam antibody
    Western Blot analysis of trkB, BDNF, Δ-FosB, Arc, synaptophysin, syntaxin-3, PSD-95 and <t>PSA-NCAM</t> in the Acb shell and Acb core of WT, HET and DAT KO rats after the microdialysis experiment. Histograms on the left are the densitometric analyses of the marker/GAPDH band gray optical density (O.D.) ratios. Blot lines on the right are representative samples of two animals from each experimental group. Values are means ± SEM of the values obtained by the reported number of rats per group. * P < 0.05 with respect to WT rats (one-way ANOVA followed by Tukey’s pairwise comparisons).
    Anti Psa Ncam Antibody, 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
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    Images

    1) Product Images from "Altered Sexual Behavior in Dopamine Transporter (DAT) Knockout Male Rats: A Behavioral, Neurochemical and Intracerebral Microdialysis Study"

    Article Title: Altered Sexual Behavior in Dopamine Transporter (DAT) Knockout Male Rats: A Behavioral, Neurochemical and Intracerebral Microdialysis Study

    Journal: Frontiers in Behavioral Neuroscience

    doi: 10.3389/fnbeh.2020.00058

    Western Blot analysis of trkB, BDNF, Δ-FosB, Arc, synaptophysin, syntaxin-3, PSD-95 and PSA-NCAM in the Acb shell and Acb core of WT, HET and DAT KO rats after the microdialysis experiment. Histograms on the left are the densitometric analyses of the marker/GAPDH band gray optical density (O.D.) ratios. Blot lines on the right are representative samples of two animals from each experimental group. Values are means ± SEM of the values obtained by the reported number of rats per group. * P < 0.05 with respect to WT rats (one-way ANOVA followed by Tukey’s pairwise comparisons).
    Figure Legend Snippet: Western Blot analysis of trkB, BDNF, Δ-FosB, Arc, synaptophysin, syntaxin-3, PSD-95 and PSA-NCAM in the Acb shell and Acb core of WT, HET and DAT KO rats after the microdialysis experiment. Histograms on the left are the densitometric analyses of the marker/GAPDH band gray optical density (O.D.) ratios. Blot lines on the right are representative samples of two animals from each experimental group. Values are means ± SEM of the values obtained by the reported number of rats per group. * P < 0.05 with respect to WT rats (one-way ANOVA followed by Tukey’s pairwise comparisons).

    Techniques Used: Western Blot, Marker

    Western Blot analysis of trkB, BDNF, Δ-FosB, Arc, synaptophysin, syntaxin-3, PSD-95 and PSA-NCAM in the mPFC and VTA of WT, HET and DAT KO rats after the microdialysis experiment. Histograms on the left are the densitometric analyses of the marker/GAPDH band gray optical density (O.D.) ratios. Blot lines on the right are representative samples of two animals from each experimental group. Values are means ± SEM of the values obtained by the reported number of rats per group. * P < 0.05, ** P < 0.01 with respect to WT rats; # P < 0.05 DAT KO with respect to HET rats (one-way ANOVA followed by Tukey’s pairwise comparisons).
    Figure Legend Snippet: Western Blot analysis of trkB, BDNF, Δ-FosB, Arc, synaptophysin, syntaxin-3, PSD-95 and PSA-NCAM in the mPFC and VTA of WT, HET and DAT KO rats after the microdialysis experiment. Histograms on the left are the densitometric analyses of the marker/GAPDH band gray optical density (O.D.) ratios. Blot lines on the right are representative samples of two animals from each experimental group. Values are means ± SEM of the values obtained by the reported number of rats per group. * P < 0.05, ** P < 0.01 with respect to WT rats; # P < 0.05 DAT KO with respect to HET rats (one-way ANOVA followed by Tukey’s pairwise comparisons).

    Techniques Used: Western Blot, Marker

    F values and significance levels of one-way ANOVAs performed on the results shown in <xref ref-type= Figures 7 , 8 ." title="F values and significance levels of one-way ANOVAs performed on the results shown ... " property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: F values and significance levels of one-way ANOVAs performed on the results shown in Figures 7 , 8 .

    Techniques Used: Marker

    rabbit anti ncam  (Alomone Labs)


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    Alomone Labs rabbit anti ncam
    Expression of <t>NCAM</t> <t>and</t> <t>profilin2</t> in the developing cerebral cortex. (A–G) Western blot analysis of NCAM and profilin2 expression in E12, E14, E16, E18, and P0 mouse cortices. γ-Tubulin served as a control. The protein levels in E14, E16, E18, and P0 mouse cortices were quantified relative to the protein levels in E12 mouse cortices set to 1.0. n = 3 or 4 biological replicates (total NCAM and profilin2, respectively). (H) Coronal sections of control and NCAM-cKO mouse cortices were coimmunostained for NCAM and Sox2 at E12 and E14. Scale bars, 20 µm. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). One-way ANOVA with least significant difference correction (C and F), with Dunnett’s T3 correction (B, D, and E), or Kruskal-Wallis test with Dunn-Bonferroni post hoc comparison (G).
    Rabbit Anti Ncam, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti ncam - by Bioz Stars, 2023-02
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    Images

    1) Product Images from "NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis"

    Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201902164

    Expression of NCAM and profilin2 in the developing cerebral cortex. (A–G) Western blot analysis of NCAM and profilin2 expression in E12, E14, E16, E18, and P0 mouse cortices. γ-Tubulin served as a control. The protein levels in E14, E16, E18, and P0 mouse cortices were quantified relative to the protein levels in E12 mouse cortices set to 1.0. n = 3 or 4 biological replicates (total NCAM and profilin2, respectively). (H) Coronal sections of control and NCAM-cKO mouse cortices were coimmunostained for NCAM and Sox2 at E12 and E14. Scale bars, 20 µm. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). One-way ANOVA with least significant difference correction (C and F), with Dunnett’s T3 correction (B, D, and E), or Kruskal-Wallis test with Dunn-Bonferroni post hoc comparison (G).
    Figure Legend Snippet: Expression of NCAM and profilin2 in the developing cerebral cortex. (A–G) Western blot analysis of NCAM and profilin2 expression in E12, E14, E16, E18, and P0 mouse cortices. γ-Tubulin served as a control. The protein levels in E14, E16, E18, and P0 mouse cortices were quantified relative to the protein levels in E12 mouse cortices set to 1.0. n = 3 or 4 biological replicates (total NCAM and profilin2, respectively). (H) Coronal sections of control and NCAM-cKO mouse cortices were coimmunostained for NCAM and Sox2 at E12 and E14. Scale bars, 20 µm. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). One-way ANOVA with least significant difference correction (C and F), with Dunnett’s T3 correction (B, D, and E), or Kruskal-Wallis test with Dunn-Bonferroni post hoc comparison (G).

    Techniques Used: Expressing, Western Blot

    Profilin2 is a novel binding partner of NCAM. (A) Coimmunoprecipitation analysis of the interaction between NCAM and profilin2 using P0 mouse brain homogenates. (B) ELISA analysis of the binding of NCAM140ICD or NCAM180ICD to immobilized profilin2. (C–E) ELISA of the binding of biotinylated NCAM140ICD-derived peptides (C), wild-type NCAM140 (aa745–753) peptide and its mutant variants with 749 LC 750 mutated to 749 AS 750 or 748 NL 749 mutated to 748 QA 749 (D), and wild-type NCAM140ICD or mutNCAM140ICD ( 749 LC 750 to 749 AS 750 mutation; E) to immobilized profilin2. n = 3 biological replicates. (F) Schematic diagram of amino acid mutations in mutNCAM140ICD. (G and H) Coronal sections of the VZ (G) and the cortex (H) of control mice were coimmunostained for profilin2, NCAM, and Sox2 (G) or Tuj1 (H). Scale bars, 50 µm. (I) Average profilin2 immunofluorescence density in each layer. (J) Percentages of profilin2 immunoreactivity in each layer. n = 9 brain slices from three mice. (K and L) Western blot analysis of levels of NCAM and profilin2 in cultured NPCs derived from E14 control and NCAM-cKO VZ/SVZ (K). The relative levels of profilin2 protein in NCAM-cKO NPCs, with the profilin2 levels in control NPCs set to 100% (L). n = 4 biological replicates. (M) Quantitative PCR analysis of the levels of profilin2 mRNA in cultured NPCs derived from E14 control and NCAM-cKO brains. Profilin2 mRNA levels in control NPCs were set to 100%. n = 5 biological replicates. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). Two-way ANOVA (B–E), one-way ANOVA with Bonferroni correction (IZ, CP, and MZ in J), Dunnett’s T3 correction (VZ/SVZ in J), Kruskal-Wallis test with Dunn-Bonferroni correction (I), and paired t test (L and M).
    Figure Legend Snippet: Profilin2 is a novel binding partner of NCAM. (A) Coimmunoprecipitation analysis of the interaction between NCAM and profilin2 using P0 mouse brain homogenates. (B) ELISA analysis of the binding of NCAM140ICD or NCAM180ICD to immobilized profilin2. (C–E) ELISA of the binding of biotinylated NCAM140ICD-derived peptides (C), wild-type NCAM140 (aa745–753) peptide and its mutant variants with 749 LC 750 mutated to 749 AS 750 or 748 NL 749 mutated to 748 QA 749 (D), and wild-type NCAM140ICD or mutNCAM140ICD ( 749 LC 750 to 749 AS 750 mutation; E) to immobilized profilin2. n = 3 biological replicates. (F) Schematic diagram of amino acid mutations in mutNCAM140ICD. (G and H) Coronal sections of the VZ (G) and the cortex (H) of control mice were coimmunostained for profilin2, NCAM, and Sox2 (G) or Tuj1 (H). Scale bars, 50 µm. (I) Average profilin2 immunofluorescence density in each layer. (J) Percentages of profilin2 immunoreactivity in each layer. n = 9 brain slices from three mice. (K and L) Western blot analysis of levels of NCAM and profilin2 in cultured NPCs derived from E14 control and NCAM-cKO VZ/SVZ (K). The relative levels of profilin2 protein in NCAM-cKO NPCs, with the profilin2 levels in control NPCs set to 100% (L). n = 4 biological replicates. (M) Quantitative PCR analysis of the levels of profilin2 mRNA in cultured NPCs derived from E14 control and NCAM-cKO brains. Profilin2 mRNA levels in control NPCs were set to 100%. n = 5 biological replicates. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). Two-way ANOVA (B–E), one-way ANOVA with Bonferroni correction (IZ, CP, and MZ in J), Dunnett’s T3 correction (VZ/SVZ in J), Kruskal-Wallis test with Dunn-Bonferroni correction (I), and paired t test (L and M).

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Mutagenesis, Immunofluorescence, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction

    Profilin2 expression is downregulated specifically by profilin2 RNAi. (A) Western blot analysis of profilin2 levels in Neuro-2a cells transfected with either siProfilin2 or NC. (B) Levels of profilin2 in siProfilin2-transfected cells relative to those in NC-transfected cells, which were set to 1.0. (C and D) Quantitative PCR analysis of the levels of profilin2 (C) or profilin1 (D) mRNA in cultured NPCs transfected with either siProfilin2 (399 or 527) or NC. The mRNA levels of profilin2/1 in NC-transfected NPCs were set to 1.0. (E and F) Western blot analysis of profilin2 levels in Neuro-2a cells transfected with scrambled shRNA (GFP) or profilin2 shRNA (shProfilin2) only or cotransfected with shProfilin2 and shRNA-resistant profilin2 (Res Profilin2). The levels of profilin2 protein were quantified relative to those in GFP-transfected cells set to 1.0. (G) NCAM levels in brain homogenates loaded in different quantities (26, 53, 78, and 104 µg). Values represent mean ± SEM. n = 4 biological replicates. *, P < 0.05; **, P < 0.01 (two sided); ns, not statistically significant. Paired t test (B), one-way ANOVA with Dunnett’s T3 correction (C and D), or least significant difference correction (F).
    Figure Legend Snippet: Profilin2 expression is downregulated specifically by profilin2 RNAi. (A) Western blot analysis of profilin2 levels in Neuro-2a cells transfected with either siProfilin2 or NC. (B) Levels of profilin2 in siProfilin2-transfected cells relative to those in NC-transfected cells, which were set to 1.0. (C and D) Quantitative PCR analysis of the levels of profilin2 (C) or profilin1 (D) mRNA in cultured NPCs transfected with either siProfilin2 (399 or 527) or NC. The mRNA levels of profilin2/1 in NC-transfected NPCs were set to 1.0. (E and F) Western blot analysis of profilin2 levels in Neuro-2a cells transfected with scrambled shRNA (GFP) or profilin2 shRNA (shProfilin2) only or cotransfected with shProfilin2 and shRNA-resistant profilin2 (Res Profilin2). The levels of profilin2 protein were quantified relative to those in GFP-transfected cells set to 1.0. (G) NCAM levels in brain homogenates loaded in different quantities (26, 53, 78, and 104 µg). Values represent mean ± SEM. n = 4 biological replicates. *, P < 0.05; **, P < 0.01 (two sided); ns, not statistically significant. Paired t test (B), one-way ANOVA with Dunnett’s T3 correction (C and D), or least significant difference correction (F).

    Techniques Used: Expressing, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Cell Culture, shRNA

    NCAM enhances NPC proliferation and differentiation through profilin2. (A) Cultured NPCs transfected with siProfilin2 or NC were incubated with NCAM antibodies and BrdU. Cells were immunostained for BrdU with DAPI counterstaining. (B, E, and F) Cultured NPCs transfected with siProfilin2 or NC were incubated with NCAM antibodies or PBS and cultured in differentiation condition for 5 d. Cells were immunostained for Tuj1 (B), GFAP (E), or O4 (F) and counterstained with DAPI. (C, D, G, and H) Percentages of BrdU + DAPI + (C), Tuj1 + DAPI + (D), GFAP + DAPI + (G), and O4 + DAPI + (H) cells in the total population of DAPI + cells. (I–K) Cultured NPCs cotransfected with profilin2 shRNA (shProfilin2) and shProfilin2-resistant plasmids (Res Profilin2), shProfilin2, or control vector expressing GFP alone (GFP) were incubated with NCAM antibodies or PBS and allowed to differentiate for 3 d. Cells were immunostained for Tuj1 or GFAP. Percentages of Tuj1 + GFP + (J) or GFAP + GFP + (K) cells in the total population of GFP + cells. n = 15 microscopic fields from three biological replicates. Scale bars, 50 µm (A, F, and I) or 20 µm (B and E). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided); ns, not statistically significant. Kruskal-Wallis test with Dunn-Bonferroni post hoc correction (C) and one-way ANOVA with Bonferroni corrections (D, G, J, and K) or Dunnett’s T3 correction (H).
    Figure Legend Snippet: NCAM enhances NPC proliferation and differentiation through profilin2. (A) Cultured NPCs transfected with siProfilin2 or NC were incubated with NCAM antibodies and BrdU. Cells were immunostained for BrdU with DAPI counterstaining. (B, E, and F) Cultured NPCs transfected with siProfilin2 or NC were incubated with NCAM antibodies or PBS and cultured in differentiation condition for 5 d. Cells were immunostained for Tuj1 (B), GFAP (E), or O4 (F) and counterstained with DAPI. (C, D, G, and H) Percentages of BrdU + DAPI + (C), Tuj1 + DAPI + (D), GFAP + DAPI + (G), and O4 + DAPI + (H) cells in the total population of DAPI + cells. (I–K) Cultured NPCs cotransfected with profilin2 shRNA (shProfilin2) and shProfilin2-resistant plasmids (Res Profilin2), shProfilin2, or control vector expressing GFP alone (GFP) were incubated with NCAM antibodies or PBS and allowed to differentiate for 3 d. Cells were immunostained for Tuj1 or GFAP. Percentages of Tuj1 + GFP + (J) or GFAP + GFP + (K) cells in the total population of GFP + cells. n = 15 microscopic fields from three biological replicates. Scale bars, 50 µm (A, F, and I) or 20 µm (B and E). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided); ns, not statistically significant. Kruskal-Wallis test with Dunn-Bonferroni post hoc correction (C) and one-way ANOVA with Bonferroni corrections (D, G, J, and K) or Dunnett’s T3 correction (H).

    Techniques Used: Cell Culture, Transfection, Incubation, shRNA, Plasmid Preparation, Expressing

    NCAM enhances NPC proliferation and differentiation through profilin2-regulated actin dynamics. (A) Western blot analysis of F- and G-actin levels in cultured control and NCAM-cKO NPCs. γ-Tubulin served as a control and was enriched in the F-actin fraction containing polymerized tubulin. (B) Relative levels of G- and F-actin in NCAM-cKO NPCs. The levels of G- and F-actin in control NPCs were set to 100%. n = 4 biological replicates. (C) Cultured MEFs were cotransfected with NCAM siRNA (siNCAM) or NC and with lentiviruses coexpressing GFP and wild-type NCAM140 (NCAM) or mutant NCAM140 (mutNCAM). MEFs cotransfected with NC and lentiviruses expressing GFP only served as a control. Western blot analysis of levels of NCAM, actin, and tubulin. Lysis with the F-actin stabilization buffer solubilizes and releases NCAM to the G-actin fraction. Relative levels of NCAM protein in the G-actin fraction and the relative ratio of G- and F-actin were quantified. n = 3 biological replicates. (D) Cultured NCAM-cKO NPCs were transduced with lentiviruses coexpressing GFP and NCAM or mutNCAM. NPCs transduced with lentiviruses expressing GFP only served as a control. NPCs were stained by fluorescent phalloidin to visualize F-actin and by DNase I to visualize G-actin. (E and F) F-actin/G-actin ratios in cells are shown in D and G, respectively. n = 54 cells (E) and 21 cells (F) from three biological replicates. (G) Cultured NCAM-cKO NPCs were transduced with plasmids coencoding either GFP or profilin2 and GFP, and then they were stained with fluorescent phalloidin and DNase I. (H) Coronal VZ sections of E12 control and NCAM-cKO mice were immunostained for actin with DAPI counterstaining. White dotted lines show examples of cell boundaries. (I) The CSI for dividing cells in the VZ. n = 40 mitotic cells from three mice. (J and K) Cultured NCAM-cKO NPCs were transduced with lentiviruses coexpressing GFP and NCAM or mutNCAM, incubated with BrdU, and immunostained for BrdU with DAPI counterstaining (J). Cultured NPCs differentiated for 5–7 d were immunostained for Tuj1 and GFAP with DAPI counterstaining (K). (L–N) Percentages of BrdU + GFP + (L), Tuj1 + GFP + (M), and GFAP + GFP + (N) cells in total GFP + cell population. n = 32 microscopic fields from three biological replicates (L). n = 5 biological replicates (M and N). Scale bars, 20 µm (D, G, J, and K) or 5 µm (H). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided); ns, not statistically significant. Paired t test (B), Mann-Whitney test (I), one-way ANOVA with Dunnett’s T3 correction (C) or Bonferroni correction (M and N), and Kruskal-Wallis test with Dunn-Bonferroni post hoc comparisons (E, F, and L).
    Figure Legend Snippet: NCAM enhances NPC proliferation and differentiation through profilin2-regulated actin dynamics. (A) Western blot analysis of F- and G-actin levels in cultured control and NCAM-cKO NPCs. γ-Tubulin served as a control and was enriched in the F-actin fraction containing polymerized tubulin. (B) Relative levels of G- and F-actin in NCAM-cKO NPCs. The levels of G- and F-actin in control NPCs were set to 100%. n = 4 biological replicates. (C) Cultured MEFs were cotransfected with NCAM siRNA (siNCAM) or NC and with lentiviruses coexpressing GFP and wild-type NCAM140 (NCAM) or mutant NCAM140 (mutNCAM). MEFs cotransfected with NC and lentiviruses expressing GFP only served as a control. Western blot analysis of levels of NCAM, actin, and tubulin. Lysis with the F-actin stabilization buffer solubilizes and releases NCAM to the G-actin fraction. Relative levels of NCAM protein in the G-actin fraction and the relative ratio of G- and F-actin were quantified. n = 3 biological replicates. (D) Cultured NCAM-cKO NPCs were transduced with lentiviruses coexpressing GFP and NCAM or mutNCAM. NPCs transduced with lentiviruses expressing GFP only served as a control. NPCs were stained by fluorescent phalloidin to visualize F-actin and by DNase I to visualize G-actin. (E and F) F-actin/G-actin ratios in cells are shown in D and G, respectively. n = 54 cells (E) and 21 cells (F) from three biological replicates. (G) Cultured NCAM-cKO NPCs were transduced with plasmids coencoding either GFP or profilin2 and GFP, and then they were stained with fluorescent phalloidin and DNase I. (H) Coronal VZ sections of E12 control and NCAM-cKO mice were immunostained for actin with DAPI counterstaining. White dotted lines show examples of cell boundaries. (I) The CSI for dividing cells in the VZ. n = 40 mitotic cells from three mice. (J and K) Cultured NCAM-cKO NPCs were transduced with lentiviruses coexpressing GFP and NCAM or mutNCAM, incubated with BrdU, and immunostained for BrdU with DAPI counterstaining (J). Cultured NPCs differentiated for 5–7 d were immunostained for Tuj1 and GFAP with DAPI counterstaining (K). (L–N) Percentages of BrdU + GFP + (L), Tuj1 + GFP + (M), and GFAP + GFP + (N) cells in total GFP + cell population. n = 32 microscopic fields from three biological replicates (L). n = 5 biological replicates (M and N). Scale bars, 20 µm (D, G, J, and K) or 5 µm (H). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided); ns, not statistically significant. Paired t test (B), Mann-Whitney test (I), one-way ANOVA with Dunnett’s T3 correction (C) or Bonferroni correction (M and N), and Kruskal-Wallis test with Dunn-Bonferroni post hoc comparisons (E, F, and L).

    Techniques Used: Western Blot, Cell Culture, Mutagenesis, Expressing, Lysis, Transduction, Staining, Incubation, MANN-WHITNEY

    The role of NCAM in regulating the temporal generation of neurons and glia in the developing cortex. (A) NCAM expression is high in NPCs at the neurogenic period and declines at the gliogenic period. The intracellular domain of NCAM interacts with profilin2 and promotes actin polymerization in NPCs. NCAM-dependent actin regulation is required for rounding of NPCs during mitosis as well as control of NPC proliferation and temporal differentiation into cortical neurons and glia. (B) Ablation of NCAM expression in NPCs results in reduced expression of profilin2 and loss of its NCAM-dependent regulation, leading to decreased actin polymerization and reduced rounding of mitotic NPCs. This slows down cell cycle progression, reduces NPC proliferation at an early stage of neural development, delays production of cortical neurons, and leads to precocious formation of cortical glia.
    Figure Legend Snippet: The role of NCAM in regulating the temporal generation of neurons and glia in the developing cortex. (A) NCAM expression is high in NPCs at the neurogenic period and declines at the gliogenic period. The intracellular domain of NCAM interacts with profilin2 and promotes actin polymerization in NPCs. NCAM-dependent actin regulation is required for rounding of NPCs during mitosis as well as control of NPC proliferation and temporal differentiation into cortical neurons and glia. (B) Ablation of NCAM expression in NPCs results in reduced expression of profilin2 and loss of its NCAM-dependent regulation, leading to decreased actin polymerization and reduced rounding of mitotic NPCs. This slows down cell cycle progression, reduces NPC proliferation at an early stage of neural development, delays production of cortical neurons, and leads to precocious formation of cortical glia.

    Techniques Used: Expressing

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    Alomone Labs rabbit anti ncam
    Expression of <t>NCAM</t> <t>and</t> <t>profilin2</t> in the developing cerebral cortex. (A–G) Western blot analysis of NCAM and profilin2 expression in E12, E14, E16, E18, and P0 mouse cortices. γ-Tubulin served as a control. The protein levels in E14, E16, E18, and P0 mouse cortices were quantified relative to the protein levels in E12 mouse cortices set to 1.0. n = 3 or 4 biological replicates (total NCAM and profilin2, respectively). (H) Coronal sections of control and NCAM-cKO mouse cortices were coimmunostained for NCAM and Sox2 at E12 and E14. Scale bars, 20 µm. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). One-way ANOVA with least significant difference correction (C and F), with Dunnett’s T3 correction (B, D, and E), or Kruskal-Wallis test with Dunn-Bonferroni post hoc comparison (G).
    Rabbit Anti Ncam, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis"

    Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201902164

    Expression of NCAM and profilin2 in the developing cerebral cortex. (A–G) Western blot analysis of NCAM and profilin2 expression in E12, E14, E16, E18, and P0 mouse cortices. γ-Tubulin served as a control. The protein levels in E14, E16, E18, and P0 mouse cortices were quantified relative to the protein levels in E12 mouse cortices set to 1.0. n = 3 or 4 biological replicates (total NCAM and profilin2, respectively). (H) Coronal sections of control and NCAM-cKO mouse cortices were coimmunostained for NCAM and Sox2 at E12 and E14. Scale bars, 20 µm. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). One-way ANOVA with least significant difference correction (C and F), with Dunnett’s T3 correction (B, D, and E), or Kruskal-Wallis test with Dunn-Bonferroni post hoc comparison (G).
    Figure Legend Snippet: Expression of NCAM and profilin2 in the developing cerebral cortex. (A–G) Western blot analysis of NCAM and profilin2 expression in E12, E14, E16, E18, and P0 mouse cortices. γ-Tubulin served as a control. The protein levels in E14, E16, E18, and P0 mouse cortices were quantified relative to the protein levels in E12 mouse cortices set to 1.0. n = 3 or 4 biological replicates (total NCAM and profilin2, respectively). (H) Coronal sections of control and NCAM-cKO mouse cortices were coimmunostained for NCAM and Sox2 at E12 and E14. Scale bars, 20 µm. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). One-way ANOVA with least significant difference correction (C and F), with Dunnett’s T3 correction (B, D, and E), or Kruskal-Wallis test with Dunn-Bonferroni post hoc comparison (G).

    Techniques Used: Expressing, Western Blot

    NCAM is dynamically expressed in NPCs during cortical development. (A and B) Coronal sections of mouse cortices from indicated embryonic stages were coimmunostained for NCAM and Sox2 (A) or Tuj1 (B). Scale bars, 50 µm. (C) Percentages of NCAM + immunoreactivity in each layer. (D) Average immunofluorescence density of NCAM in each layer. n = 9 brain slices from three mice. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). One-way ANOVA with Bonferroni corrections (IZ, CP, and MZ in C and MZ in D), Dunnett’s T3 correction (VZ/SVZ in C), and Kruskal-Wallis test with Dunn-Bonferroni correction (VZ/SVZ, IZ, and CP in D). PP, preplate.
    Figure Legend Snippet: NCAM is dynamically expressed in NPCs during cortical development. (A and B) Coronal sections of mouse cortices from indicated embryonic stages were coimmunostained for NCAM and Sox2 (A) or Tuj1 (B). Scale bars, 50 µm. (C) Percentages of NCAM + immunoreactivity in each layer. (D) Average immunofluorescence density of NCAM in each layer. n = 9 brain slices from three mice. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). One-way ANOVA with Bonferroni corrections (IZ, CP, and MZ in C and MZ in D), Dunnett’s T3 correction (VZ/SVZ in C), and Kruskal-Wallis test with Dunn-Bonferroni correction (VZ/SVZ, IZ, and CP in D). PP, preplate.

    Techniques Used: Immunofluorescence

    Profilin2 is a novel binding partner of NCAM. (A) Coimmunoprecipitation analysis of the interaction between NCAM and profilin2 using P0 mouse brain homogenates. (B) ELISA analysis of the binding of NCAM140ICD or NCAM180ICD to immobilized profilin2. (C–E) ELISA of the binding of biotinylated NCAM140ICD-derived peptides (C), wild-type NCAM140 (aa745–753) peptide and its mutant variants with 749 LC 750 mutated to 749 AS 750 or 748 NL 749 mutated to 748 QA 749 (D), and wild-type NCAM140ICD or mutNCAM140ICD ( 749 LC 750 to 749 AS 750 mutation; E) to immobilized profilin2. n = 3 biological replicates. (F) Schematic diagram of amino acid mutations in mutNCAM140ICD. (G and H) Coronal sections of the VZ (G) and the cortex (H) of control mice were coimmunostained for profilin2, NCAM, and Sox2 (G) or Tuj1 (H). Scale bars, 50 µm. (I) Average profilin2 immunofluorescence density in each layer. (J) Percentages of profilin2 immunoreactivity in each layer. n = 9 brain slices from three mice. (K and L) Western blot analysis of levels of NCAM and profilin2 in cultured NPCs derived from E14 control and NCAM-cKO VZ/SVZ (K). The relative levels of profilin2 protein in NCAM-cKO NPCs, with the profilin2 levels in control NPCs set to 100% (L). n = 4 biological replicates. (M) Quantitative PCR analysis of the levels of profilin2 mRNA in cultured NPCs derived from E14 control and NCAM-cKO brains. Profilin2 mRNA levels in control NPCs were set to 100%. n = 5 biological replicates. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). Two-way ANOVA (B–E), one-way ANOVA with Bonferroni correction (IZ, CP, and MZ in J), Dunnett’s T3 correction (VZ/SVZ in J), Kruskal-Wallis test with Dunn-Bonferroni correction (I), and paired t test (L and M).
    Figure Legend Snippet: Profilin2 is a novel binding partner of NCAM. (A) Coimmunoprecipitation analysis of the interaction between NCAM and profilin2 using P0 mouse brain homogenates. (B) ELISA analysis of the binding of NCAM140ICD or NCAM180ICD to immobilized profilin2. (C–E) ELISA of the binding of biotinylated NCAM140ICD-derived peptides (C), wild-type NCAM140 (aa745–753) peptide and its mutant variants with 749 LC 750 mutated to 749 AS 750 or 748 NL 749 mutated to 748 QA 749 (D), and wild-type NCAM140ICD or mutNCAM140ICD ( 749 LC 750 to 749 AS 750 mutation; E) to immobilized profilin2. n = 3 biological replicates. (F) Schematic diagram of amino acid mutations in mutNCAM140ICD. (G and H) Coronal sections of the VZ (G) and the cortex (H) of control mice were coimmunostained for profilin2, NCAM, and Sox2 (G) or Tuj1 (H). Scale bars, 50 µm. (I) Average profilin2 immunofluorescence density in each layer. (J) Percentages of profilin2 immunoreactivity in each layer. n = 9 brain slices from three mice. (K and L) Western blot analysis of levels of NCAM and profilin2 in cultured NPCs derived from E14 control and NCAM-cKO VZ/SVZ (K). The relative levels of profilin2 protein in NCAM-cKO NPCs, with the profilin2 levels in control NPCs set to 100% (L). n = 4 biological replicates. (M) Quantitative PCR analysis of the levels of profilin2 mRNA in cultured NPCs derived from E14 control and NCAM-cKO brains. Profilin2 mRNA levels in control NPCs were set to 100%. n = 5 biological replicates. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). Two-way ANOVA (B–E), one-way ANOVA with Bonferroni correction (IZ, CP, and MZ in J), Dunnett’s T3 correction (VZ/SVZ in J), Kruskal-Wallis test with Dunn-Bonferroni correction (I), and paired t test (L and M).

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Mutagenesis, Immunofluorescence, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction

    Profilin2 expression is downregulated specifically by profilin2 RNAi. (A) Western blot analysis of profilin2 levels in Neuro-2a cells transfected with either siProfilin2 or NC. (B) Levels of profilin2 in siProfilin2-transfected cells relative to those in NC-transfected cells, which were set to 1.0. (C and D) Quantitative PCR analysis of the levels of profilin2 (C) or profilin1 (D) mRNA in cultured NPCs transfected with either siProfilin2 (399 or 527) or NC. The mRNA levels of profilin2/1 in NC-transfected NPCs were set to 1.0. (E and F) Western blot analysis of profilin2 levels in Neuro-2a cells transfected with scrambled shRNA (GFP) or profilin2 shRNA (shProfilin2) only or cotransfected with shProfilin2 and shRNA-resistant profilin2 (Res Profilin2). The levels of profilin2 protein were quantified relative to those in GFP-transfected cells set to 1.0. (G) NCAM levels in brain homogenates loaded in different quantities (26, 53, 78, and 104 µg). Values represent mean ± SEM. n = 4 biological replicates. *, P < 0.05; **, P < 0.01 (two sided); ns, not statistically significant. Paired t test (B), one-way ANOVA with Dunnett’s T3 correction (C and D), or least significant difference correction (F).
    Figure Legend Snippet: Profilin2 expression is downregulated specifically by profilin2 RNAi. (A) Western blot analysis of profilin2 levels in Neuro-2a cells transfected with either siProfilin2 or NC. (B) Levels of profilin2 in siProfilin2-transfected cells relative to those in NC-transfected cells, which were set to 1.0. (C and D) Quantitative PCR analysis of the levels of profilin2 (C) or profilin1 (D) mRNA in cultured NPCs transfected with either siProfilin2 (399 or 527) or NC. The mRNA levels of profilin2/1 in NC-transfected NPCs were set to 1.0. (E and F) Western blot analysis of profilin2 levels in Neuro-2a cells transfected with scrambled shRNA (GFP) or profilin2 shRNA (shProfilin2) only or cotransfected with shProfilin2 and shRNA-resistant profilin2 (Res Profilin2). The levels of profilin2 protein were quantified relative to those in GFP-transfected cells set to 1.0. (G) NCAM levels in brain homogenates loaded in different quantities (26, 53, 78, and 104 µg). Values represent mean ± SEM. n = 4 biological replicates. *, P < 0.05; **, P < 0.01 (two sided); ns, not statistically significant. Paired t test (B), one-way ANOVA with Dunnett’s T3 correction (C and D), or least significant difference correction (F).

    Techniques Used: Expressing, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Cell Culture, shRNA

    NCAM enhances NPC proliferation and differentiation through profilin2. (A) Cultured NPCs transfected with siProfilin2 or NC were incubated with NCAM antibodies and BrdU. Cells were immunostained for BrdU with DAPI counterstaining. (B, E, and F) Cultured NPCs transfected with siProfilin2 or NC were incubated with NCAM antibodies or PBS and cultured in differentiation condition for 5 d. Cells were immunostained for Tuj1 (B), GFAP (E), or O4 (F) and counterstained with DAPI. (C, D, G, and H) Percentages of BrdU + DAPI + (C), Tuj1 + DAPI + (D), GFAP + DAPI + (G), and O4 + DAPI + (H) cells in the total population of DAPI + cells. (I–K) Cultured NPCs cotransfected with profilin2 shRNA (shProfilin2) and shProfilin2-resistant plasmids (Res Profilin2), shProfilin2, or control vector expressing GFP alone (GFP) were incubated with NCAM antibodies or PBS and allowed to differentiate for 3 d. Cells were immunostained for Tuj1 or GFAP. Percentages of Tuj1 + GFP + (J) or GFAP + GFP + (K) cells in the total population of GFP + cells. n = 15 microscopic fields from three biological replicates. Scale bars, 50 µm (A, F, and I) or 20 µm (B and E). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided); ns, not statistically significant. Kruskal-Wallis test with Dunn-Bonferroni post hoc correction (C) and one-way ANOVA with Bonferroni corrections (D, G, J, and K) or Dunnett’s T3 correction (H).
    Figure Legend Snippet: NCAM enhances NPC proliferation and differentiation through profilin2. (A) Cultured NPCs transfected with siProfilin2 or NC were incubated with NCAM antibodies and BrdU. Cells were immunostained for BrdU with DAPI counterstaining. (B, E, and F) Cultured NPCs transfected with siProfilin2 or NC were incubated with NCAM antibodies or PBS and cultured in differentiation condition for 5 d. Cells were immunostained for Tuj1 (B), GFAP (E), or O4 (F) and counterstained with DAPI. (C, D, G, and H) Percentages of BrdU + DAPI + (C), Tuj1 + DAPI + (D), GFAP + DAPI + (G), and O4 + DAPI + (H) cells in the total population of DAPI + cells. (I–K) Cultured NPCs cotransfected with profilin2 shRNA (shProfilin2) and shProfilin2-resistant plasmids (Res Profilin2), shProfilin2, or control vector expressing GFP alone (GFP) were incubated with NCAM antibodies or PBS and allowed to differentiate for 3 d. Cells were immunostained for Tuj1 or GFAP. Percentages of Tuj1 + GFP + (J) or GFAP + GFP + (K) cells in the total population of GFP + cells. n = 15 microscopic fields from three biological replicates. Scale bars, 50 µm (A, F, and I) or 20 µm (B and E). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided); ns, not statistically significant. Kruskal-Wallis test with Dunn-Bonferroni post hoc correction (C) and one-way ANOVA with Bonferroni corrections (D, G, J, and K) or Dunnett’s T3 correction (H).

    Techniques Used: Cell Culture, Transfection, Incubation, shRNA, Plasmid Preparation, Expressing

    NCAM enhances NPC proliferation and differentiation through profilin2-regulated actin dynamics. (A) Western blot analysis of F- and G-actin levels in cultured control and NCAM-cKO NPCs. γ-Tubulin served as a control and was enriched in the F-actin fraction containing polymerized tubulin. (B) Relative levels of G- and F-actin in NCAM-cKO NPCs. The levels of G- and F-actin in control NPCs were set to 100%. n = 4 biological replicates. (C) Cultured MEFs were cotransfected with NCAM siRNA (siNCAM) or NC and with lentiviruses coexpressing GFP and wild-type NCAM140 (NCAM) or mutant NCAM140 (mutNCAM). MEFs cotransfected with NC and lentiviruses expressing GFP only served as a control. Western blot analysis of levels of NCAM, actin, and tubulin. Lysis with the F-actin stabilization buffer solubilizes and releases NCAM to the G-actin fraction. Relative levels of NCAM protein in the G-actin fraction and the relative ratio of G- and F-actin were quantified. n = 3 biological replicates. (D) Cultured NCAM-cKO NPCs were transduced with lentiviruses coexpressing GFP and NCAM or mutNCAM. NPCs transduced with lentiviruses expressing GFP only served as a control. NPCs were stained by fluorescent phalloidin to visualize F-actin and by DNase I to visualize G-actin. (E and F) F-actin/G-actin ratios in cells are shown in D and G, respectively. n = 54 cells (E) and 21 cells (F) from three biological replicates. (G) Cultured NCAM-cKO NPCs were transduced with plasmids coencoding either GFP or profilin2 and GFP, and then they were stained with fluorescent phalloidin and DNase I. (H) Coronal VZ sections of E12 control and NCAM-cKO mice were immunostained for actin with DAPI counterstaining. White dotted lines show examples of cell boundaries. (I) The CSI for dividing cells in the VZ. n = 40 mitotic cells from three mice. (J and K) Cultured NCAM-cKO NPCs were transduced with lentiviruses coexpressing GFP and NCAM or mutNCAM, incubated with BrdU, and immunostained for BrdU with DAPI counterstaining (J). Cultured NPCs differentiated for 5–7 d were immunostained for Tuj1 and GFAP with DAPI counterstaining (K). (L–N) Percentages of BrdU + GFP + (L), Tuj1 + GFP + (M), and GFAP + GFP + (N) cells in total GFP + cell population. n = 32 microscopic fields from three biological replicates (L). n = 5 biological replicates (M and N). Scale bars, 20 µm (D, G, J, and K) or 5 µm (H). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided); ns, not statistically significant. Paired t test (B), Mann-Whitney test (I), one-way ANOVA with Dunnett’s T3 correction (C) or Bonferroni correction (M and N), and Kruskal-Wallis test with Dunn-Bonferroni post hoc comparisons (E, F, and L).
    Figure Legend Snippet: NCAM enhances NPC proliferation and differentiation through profilin2-regulated actin dynamics. (A) Western blot analysis of F- and G-actin levels in cultured control and NCAM-cKO NPCs. γ-Tubulin served as a control and was enriched in the F-actin fraction containing polymerized tubulin. (B) Relative levels of G- and F-actin in NCAM-cKO NPCs. The levels of G- and F-actin in control NPCs were set to 100%. n = 4 biological replicates. (C) Cultured MEFs were cotransfected with NCAM siRNA (siNCAM) or NC and with lentiviruses coexpressing GFP and wild-type NCAM140 (NCAM) or mutant NCAM140 (mutNCAM). MEFs cotransfected with NC and lentiviruses expressing GFP only served as a control. Western blot analysis of levels of NCAM, actin, and tubulin. Lysis with the F-actin stabilization buffer solubilizes and releases NCAM to the G-actin fraction. Relative levels of NCAM protein in the G-actin fraction and the relative ratio of G- and F-actin were quantified. n = 3 biological replicates. (D) Cultured NCAM-cKO NPCs were transduced with lentiviruses coexpressing GFP and NCAM or mutNCAM. NPCs transduced with lentiviruses expressing GFP only served as a control. NPCs were stained by fluorescent phalloidin to visualize F-actin and by DNase I to visualize G-actin. (E and F) F-actin/G-actin ratios in cells are shown in D and G, respectively. n = 54 cells (E) and 21 cells (F) from three biological replicates. (G) Cultured NCAM-cKO NPCs were transduced with plasmids coencoding either GFP or profilin2 and GFP, and then they were stained with fluorescent phalloidin and DNase I. (H) Coronal VZ sections of E12 control and NCAM-cKO mice were immunostained for actin with DAPI counterstaining. White dotted lines show examples of cell boundaries. (I) The CSI for dividing cells in the VZ. n = 40 mitotic cells from three mice. (J and K) Cultured NCAM-cKO NPCs were transduced with lentiviruses coexpressing GFP and NCAM or mutNCAM, incubated with BrdU, and immunostained for BrdU with DAPI counterstaining (J). Cultured NPCs differentiated for 5–7 d were immunostained for Tuj1 and GFAP with DAPI counterstaining (K). (L–N) Percentages of BrdU + GFP + (L), Tuj1 + GFP + (M), and GFAP + GFP + (N) cells in total GFP + cell population. n = 32 microscopic fields from three biological replicates (L). n = 5 biological replicates (M and N). Scale bars, 20 µm (D, G, J, and K) or 5 µm (H). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided); ns, not statistically significant. Paired t test (B), Mann-Whitney test (I), one-way ANOVA with Dunnett’s T3 correction (C) or Bonferroni correction (M and N), and Kruskal-Wallis test with Dunn-Bonferroni post hoc comparisons (E, F, and L).

    Techniques Used: Western Blot, Cell Culture, Mutagenesis, Expressing, Lysis, Transduction, Staining, Incubation, MANN-WHITNEY

    The role of NCAM in regulating the temporal generation of neurons and glia in the developing cortex. (A) NCAM expression is high in NPCs at the neurogenic period and declines at the gliogenic period. The intracellular domain of NCAM interacts with profilin2 and promotes actin polymerization in NPCs. NCAM-dependent actin regulation is required for rounding of NPCs during mitosis as well as control of NPC proliferation and temporal differentiation into cortical neurons and glia. (B) Ablation of NCAM expression in NPCs results in reduced expression of profilin2 and loss of its NCAM-dependent regulation, leading to decreased actin polymerization and reduced rounding of mitotic NPCs. This slows down cell cycle progression, reduces NPC proliferation at an early stage of neural development, delays production of cortical neurons, and leads to precocious formation of cortical glia.
    Figure Legend Snippet: The role of NCAM in regulating the temporal generation of neurons and glia in the developing cortex. (A) NCAM expression is high in NPCs at the neurogenic period and declines at the gliogenic period. The intracellular domain of NCAM interacts with profilin2 and promotes actin polymerization in NPCs. NCAM-dependent actin regulation is required for rounding of NPCs during mitosis as well as control of NPC proliferation and temporal differentiation into cortical neurons and glia. (B) Ablation of NCAM expression in NPCs results in reduced expression of profilin2 and loss of its NCAM-dependent regulation, leading to decreased actin polymerization and reduced rounding of mitotic NPCs. This slows down cell cycle progression, reduces NPC proliferation at an early stage of neural development, delays production of cortical neurons, and leads to precocious formation of cortical glia.

    Techniques Used: Expressing

    anti psa ncam antibody  (Alomone Labs)


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    Alomone Labs anti psa ncam antibody
    Western blot analysis of the frontal cortex homogenates in vehicle-treated and resveratrol (RVT)-treated rats either in sham-operated or after bilateral common carotid artery occlusion followed by reperfusion (BCCAO/R). ( A , B ) Brain-derived neurotrophic factor (BDNF), ( C , D ) tyrosine kinase receptor B (trkB), ( E , F ) polysialylated neural cell-adhesion molecule <t>(PSA-NCAM),</t> and ( G , H ) activity-regulated cytoskeleton-associated (Arc) protein. ( B , D , F , H ) Densitometric analysis of the band gray levels expressed as a percentage of the optical density (O.D.) ratio of BDNF-, trkB-, PSA-NCAM-, and Arc-positive bands to those immunostained for GAPDH. Data are reported as the mean values of 12 vehicle-treated and 10 RVT-treated rats. Error bars depict the standard error of the mean (S.E.M.). Asterisks denote significant differences. Two-way ANOVA with the Tukey’s test for post hoc analyses was applied to evaluate statistical differences between groups. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (see for F - and p -values relevant to effects of BCCAO/R and RVT pre-treatment and to the interaction between them).
    Anti Psa Ncam Antibody, 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
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    1) Product Images from "Resveratrol Regulates BDNF, trkB, PSA-NCAM, and Arc Expression in the Rat Cerebral Cortex after Bilateral Common Carotid Artery Occlusion and Reperfusion"

    Article Title: Resveratrol Regulates BDNF, trkB, PSA-NCAM, and Arc Expression in the Rat Cerebral Cortex after Bilateral Common Carotid Artery Occlusion and Reperfusion

    Journal: Nutrients

    doi: 10.3390/nu11051000

    Western blot analysis of the frontal cortex homogenates in vehicle-treated and resveratrol (RVT)-treated rats either in sham-operated or after bilateral common carotid artery occlusion followed by reperfusion (BCCAO/R). ( A , B ) Brain-derived neurotrophic factor (BDNF), ( C , D ) tyrosine kinase receptor B (trkB), ( E , F ) polysialylated neural cell-adhesion molecule (PSA-NCAM), and ( G , H ) activity-regulated cytoskeleton-associated (Arc) protein. ( B , D , F , H ) Densitometric analysis of the band gray levels expressed as a percentage of the optical density (O.D.) ratio of BDNF-, trkB-, PSA-NCAM-, and Arc-positive bands to those immunostained for GAPDH. Data are reported as the mean values of 12 vehicle-treated and 10 RVT-treated rats. Error bars depict the standard error of the mean (S.E.M.). Asterisks denote significant differences. Two-way ANOVA with the Tukey’s test for post hoc analyses was applied to evaluate statistical differences between groups. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (see for F - and p -values relevant to effects of BCCAO/R and RVT pre-treatment and to the interaction between them).
    Figure Legend Snippet: Western blot analysis of the frontal cortex homogenates in vehicle-treated and resveratrol (RVT)-treated rats either in sham-operated or after bilateral common carotid artery occlusion followed by reperfusion (BCCAO/R). ( A , B ) Brain-derived neurotrophic factor (BDNF), ( C , D ) tyrosine kinase receptor B (trkB), ( E , F ) polysialylated neural cell-adhesion molecule (PSA-NCAM), and ( G , H ) activity-regulated cytoskeleton-associated (Arc) protein. ( B , D , F , H ) Densitometric analysis of the band gray levels expressed as a percentage of the optical density (O.D.) ratio of BDNF-, trkB-, PSA-NCAM-, and Arc-positive bands to those immunostained for GAPDH. Data are reported as the mean values of 12 vehicle-treated and 10 RVT-treated rats. Error bars depict the standard error of the mean (S.E.M.). Asterisks denote significant differences. Two-way ANOVA with the Tukey’s test for post hoc analyses was applied to evaluate statistical differences between groups. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (see for F - and p -values relevant to effects of BCCAO/R and RVT pre-treatment and to the interaction between them).

    Techniques Used: Western Blot, Derivative Assay, Activity Assay

    F -values and significance levels from two-way ANOVA performed on data obtained after BCCAO/R and resveratrol (RVT) pre-treatment by means of Western blot in the rat frontal cortex.
    Figure Legend Snippet: F -values and significance levels from two-way ANOVA performed on data obtained after BCCAO/R and resveratrol (RVT) pre-treatment by means of Western blot in the rat frontal cortex.

    Techniques Used: Western Blot

    anti psa ncam antibody  (Alomone Labs)


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    Alomone Labs anti psa ncam antibody
    Western blot analysis of the brain-derived neurotrophic factor (BDNF) ( A , B ), trkB ( C , D ), and the polysialilated-neural cell adhesion molecule <t>(PSA-NCAM)</t> ( E , F ) in the dorsal hippocampus of the RHA and the RLA rats, under the baseline conditions (CONTROL), and after forced swimming (FS). ( A , C , E ): BDNF-immunostained blots ( A ), trkB-immunostained blots ( C ), and PSA-NCAM-immunostained blots (E), showing representative samples from two rats; ( B , D , F ): Densitometric analysis of the BDNF/GAPDH (B), trkB/GAPDH (D), and the PSA-NCAM/GAPDH band gray optical density (O.D.) ratios (F). Columns and bars denote the mean ± S.E.M. of eight rats in each experimental group. *: p < 0.05. (Tukey’s post hoc test for, multiple comparisons).
    Anti Psa Ncam Antibody, 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
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    1) Product Images from "Effect of Acute Stress on the Expression of BDNF, trkB, and PSA-NCAM in the Hippocampus of the Roman Rats: A Genetic Model of Vulnerability/Resistance to Stress-Induced Depression"

    Article Title: Effect of Acute Stress on the Expression of BDNF, trkB, and PSA-NCAM in the Hippocampus of the Roman Rats: A Genetic Model of Vulnerability/Resistance to Stress-Induced Depression

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19123745

    Western blot analysis of the brain-derived neurotrophic factor (BDNF) ( A , B ), trkB ( C , D ), and the polysialilated-neural cell adhesion molecule (PSA-NCAM) ( E , F ) in the dorsal hippocampus of the RHA and the RLA rats, under the baseline conditions (CONTROL), and after forced swimming (FS). ( A , C , E ): BDNF-immunostained blots ( A ), trkB-immunostained blots ( C ), and PSA-NCAM-immunostained blots (E), showing representative samples from two rats; ( B , D , F ): Densitometric analysis of the BDNF/GAPDH (B), trkB/GAPDH (D), and the PSA-NCAM/GAPDH band gray optical density (O.D.) ratios (F). Columns and bars denote the mean ± S.E.M. of eight rats in each experimental group. *: p < 0.05. (Tukey’s post hoc test for, multiple comparisons).
    Figure Legend Snippet: Western blot analysis of the brain-derived neurotrophic factor (BDNF) ( A , B ), trkB ( C , D ), and the polysialilated-neural cell adhesion molecule (PSA-NCAM) ( E , F ) in the dorsal hippocampus of the RHA and the RLA rats, under the baseline conditions (CONTROL), and after forced swimming (FS). ( A , C , E ): BDNF-immunostained blots ( A ), trkB-immunostained blots ( C ), and PSA-NCAM-immunostained blots (E), showing representative samples from two rats; ( B , D , F ): Densitometric analysis of the BDNF/GAPDH (B), trkB/GAPDH (D), and the PSA-NCAM/GAPDH band gray optical density (O.D.) ratios (F). Columns and bars denote the mean ± S.E.M. of eight rats in each experimental group. *: p < 0.05. (Tukey’s post hoc test for, multiple comparisons).

    Techniques Used: Western Blot, Derivative Assay

    Western blot analysis of the BDNF ( A , B ), trkB ( C , D ), and the PSA-NCAM ( E , F ), in the ventral hippocampus of the RHA and the RLA rats, under the baseline conditions (CONTROL), and after forced swimming (FS). ( A , C , E ): BDNF-immunostained blots ( A ), trkB-immunostained blots ( C ), and PSA-NCAM-immunostained blots ( E ) showing representative samples from two rats; ( B , D , F ): Densitometric analysis of the BDNF/GAPDH ( B ), trkB/GAPDH ( D ), and the PSA-NCAM/GAPDH band gray optical density (O.D.) ratios ( F ). The columns and bars denote the mean ± S.E.M. of eight rats, in each experimental group. *: p < 0.05; **: p < 0.01 (Tukey’s post hoc test or Sidak’s correction, for multiple comparisons).
    Figure Legend Snippet: Western blot analysis of the BDNF ( A , B ), trkB ( C , D ), and the PSA-NCAM ( E , F ), in the ventral hippocampus of the RHA and the RLA rats, under the baseline conditions (CONTROL), and after forced swimming (FS). ( A , C , E ): BDNF-immunostained blots ( A ), trkB-immunostained blots ( C ), and PSA-NCAM-immunostained blots ( E ) showing representative samples from two rats; ( B , D , F ): Densitometric analysis of the BDNF/GAPDH ( B ), trkB/GAPDH ( D ), and the PSA-NCAM/GAPDH band gray optical density (O.D.) ratios ( F ). The columns and bars denote the mean ± S.E.M. of eight rats, in each experimental group. *: p < 0.05; **: p < 0.01 (Tukey’s post hoc test or Sidak’s correction, for multiple comparisons).

    Techniques Used: Western Blot

    F values and significance levels of two-way ANOVAs performed on western blot data, shown in <xref ref-type= Figure 2 and Figure 3 ." title="F values and significance levels of two-way ANOVAs performed on western blot data, ... " property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: F values and significance levels of two-way ANOVAs performed on western blot data, shown in Figure 2 and Figure 3 .

    Techniques Used: Western Blot, Marker

    F values and significance levels of two-way ANOVAs performed on data obtained from the densitometric analysis of tissue section distribution of the BDNF- like immunoreactivity (LI), the trkB-LI, and the  PSA-NCAM-LI,  shown in <xref ref-type= Figures S1–S6 ." title="... BDNF- like immunoreactivity (LI), the trkB-LI, and the PSA-NCAM-LI, shown in Figures ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: F values and significance levels of two-way ANOVAs performed on data obtained from the densitometric analysis of tissue section distribution of the BDNF- like immunoreactivity (LI), the trkB-LI, and the PSA-NCAM-LI, shown in Figures S1–S6 .

    Techniques Used: Marker

    Densitometric analysis of the PSA-NCAM-like immunoreactivity in the CA1–CA3 sectors of the Ammon’s horn and in the dentate gyrus (DG) of the dorsal hippocampus, in baseline conditions (CONTROL) and after forced swimming (FS). Columns and bars denote the mean ± S.E.M. of six rats, in each experimental group. Two different sections were analyzed for each rat. *: p < 0.05; **: p < 0.01 (Tukey’s post hoc test or Sidak’s correction for multiple comparisons).
    Figure Legend Snippet: Densitometric analysis of the PSA-NCAM-like immunoreactivity in the CA1–CA3 sectors of the Ammon’s horn and in the dentate gyrus (DG) of the dorsal hippocampus, in baseline conditions (CONTROL) and after forced swimming (FS). Columns and bars denote the mean ± S.E.M. of six rats, in each experimental group. Two different sections were analyzed for each rat. *: p < 0.05; **: p < 0.01 (Tukey’s post hoc test or Sidak’s correction for multiple comparisons).

    Techniques Used:

    Densitometric analysis of the PSA-NCAM-like immunoreactivity in the CA1 and CA3 sectors of the Ammon’s horn and in the dentate gyrus (DG) of the ventral hippocampus, in baseline conditions (CONTROL) and after forced swimming (FS). Columns and bars denote the mean ± S.E.M. of six rats, in each experimental group. Two different sections were analyzed for each rat. *: p < 0.05; ** p < 0.01; ****: p < 0.0001 (Tukey’s post hoc test or Sidak’s correction for multiple comparisons).
    Figure Legend Snippet: Densitometric analysis of the PSA-NCAM-like immunoreactivity in the CA1 and CA3 sectors of the Ammon’s horn and in the dentate gyrus (DG) of the ventral hippocampus, in baseline conditions (CONTROL) and after forced swimming (FS). Columns and bars denote the mean ± S.E.M. of six rats, in each experimental group. Two different sections were analyzed for each rat. *: p < 0.05; ** p < 0.01; ****: p < 0.0001 (Tukey’s post hoc test or Sidak’s correction for multiple comparisons).

    Techniques Used:

    alexa fluor 488 goat anti rabbit igg  (Alomone Labs)


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    Alomone Labs alexa fluor 488 goat anti rabbit igg
    Alexa Fluor 488 Goat Anti Rabbit Igg, 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
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    alexa fluor 488 goat anti rabbit igg  (Alomone Labs)


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    Alomone Labs alexa fluor 488 goat anti rabbit igg
    Alexa Fluor 488 Goat Anti Rabbit Igg, 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/alexa fluor 488 goat anti rabbit igg/product/Alomone Labs
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    Alomone Labs ncam 1
    NCAM -1 and ALCAM are enriched in the murine VCS. (A) Scatter plots identified <t>Ncam-1</t> and Alcam (red) as two of the most enriched genes with cell adhesion properties in mature PCs. (B) Quantitative RT-PCR (qPCR) confirmed significant Ncam-1 and Alcam enrichment in adult PCs in addition to other known markers of the VCS ( Cx40 and Cntn2 ) ( n =3). Error bars represent s.e.m. (C) Immunofluorescence staining in WT P21 murine hearts sections in the VCS shows overlapping expression of NCAM-1 (red) and ALCAM (red) with Cx40 (green) in the HPS. Scale bars: 25 μm.
    Ncam 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ncam 1 - by Bioz Stars, 2023-02
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    93
    Alomone Labs ncam
    (A) RT-PCR <t>for</t> <t>trpm8</t> (236 bp) from cDNA extracted from stage 24 whole embryo or dissected spinal cord. +/− RT, in the presence or absence of the reverse transcriptase, respectively, during conversion of isolated mRNA into cDNA. (B) In situ hybridization for trpm8 in stage 24 embryos showing specific labeling in brain and spinal cord. (C) Western blot assays from egg, stage 24 wild-type whole embryo or from stage 40 whole larva, control morpholino (MO) or TRPM8-translation-blockingMO 1 (TRPM8-tbMO1) lysates. Predicted TRPM8 molecular weight [MW]: 132 kDa. Shown are representative examples of one of 3 independent experiments. β-tubulin was used as loading control. (D) Immunostained transverse section of stage 25 spinal cord (outlined). D, dorsal; V, ventral; scale bar, 20 μm; arrows indicate TRPM8 clusters in ventral neuron domains. <t>NCAM</t> labeling was used as counterstaining. (E) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged at 1 Hz for 90 s. Either 100 μM (−)-menthol or vehicle (0.05% DMSO)was added after 35 s of imaging and recording continued for another 60 s. Images show a menthol-responsive ventral neuron before (left, control) and after (right) addition of (−)-menthol. Colored scale shows fluorescence intensity in arbitrary units. Traces show the changes in fluorescence for the indicated cell (arrow) in both trials. (F) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged in 30-min intervals at cold (14.5°C) and warm (26.5°C) temperatures in the absence (vehicle, 0.1% DMSO) or presence of 10 μM AMTB, TRPM8 inhibitor. Scatterplots show changes in Ca 2+ spike frequency when switching temperatures in individual spinal neurons and geometric mean (black lines) from N = 3 ventral spinal cords per condition (n of neurons analyzed: DMSO, 62; AMTB, 71). Teal circles represent neurons with higher spike frequency at 14.5°C, magenta circles represent neurons with higher spike frequency at 26.5°C, and black circles represent neurons with no change in spike frequency across temperatures; ****p < 0.0001, comparison within treatments Wilcoxon matched-pairs signed rank, two-tailed test. (G) RT-PCR from cDNA collected from stage 46 larvae previously injected with 2.5 pmol standard control morpholino (Control-MO) or TRPM8-splicing-blocking morpholino (TRPM8-sbMO) shows that trpm8 mature transcript (349 bp) is not detected in TRPM8-sbMO animals. odc : ornithine decarboxylase (101 bp) as positive control. (H) TRPM8-sbMO or Control-MO containing spinal cord from stage 24 embryos were Ca 2+ -imaged for 30 min at cold temperature (14.5°C). Graph shows individual (scatterplots) and geometric mean (black lines) Ca 2+ spike frequency from N = 3 ventral spinal cords pergroup (n of neurons analyzed: Control-MO, 81; TRPM8-sbMO, 64), ****p < 0.001, Kolmogorov-Smirnov, two-tailed test. See also and .
    Ncam, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs anti psa ncam antibody
    Western Blot analysis of trkB, BDNF, Δ-FosB, Arc, synaptophysin, syntaxin-3, PSD-95 and <t>PSA-NCAM</t> in the Acb shell and Acb core of WT, HET and DAT KO rats after the microdialysis experiment. Histograms on the left are the densitometric analyses of the marker/GAPDH band gray optical density (O.D.) ratios. Blot lines on the right are representative samples of two animals from each experimental group. Values are means ± SEM of the values obtained by the reported number of rats per group. * P < 0.05 with respect to WT rats (one-way ANOVA followed by Tukey’s pairwise comparisons).
    Anti Psa Ncam Antibody, 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
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    Alomone Labs rabbit anti ncam
    Expression of <t>NCAM</t> <t>and</t> <t>profilin2</t> in the developing cerebral cortex. (A–G) Western blot analysis of NCAM and profilin2 expression in E12, E14, E16, E18, and P0 mouse cortices. γ-Tubulin served as a control. The protein levels in E14, E16, E18, and P0 mouse cortices were quantified relative to the protein levels in E12 mouse cortices set to 1.0. n = 3 or 4 biological replicates (total NCAM and profilin2, respectively). (H) Coronal sections of control and NCAM-cKO mouse cortices were coimmunostained for NCAM and Sox2 at E12 and E14. Scale bars, 20 µm. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). One-way ANOVA with least significant difference correction (C and F), with Dunnett’s T3 correction (B, D, and E), or Kruskal-Wallis test with Dunn-Bonferroni post hoc comparison (G).
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    Alomone Labs alexa fluor 488 goat anti rabbit igg
    Expression of <t>NCAM</t> <t>and</t> <t>profilin2</t> in the developing cerebral cortex. (A–G) Western blot analysis of NCAM and profilin2 expression in E12, E14, E16, E18, and P0 mouse cortices. γ-Tubulin served as a control. The protein levels in E14, E16, E18, and P0 mouse cortices were quantified relative to the protein levels in E12 mouse cortices set to 1.0. n = 3 or 4 biological replicates (total NCAM and profilin2, respectively). (H) Coronal sections of control and NCAM-cKO mouse cortices were coimmunostained for NCAM and Sox2 at E12 and E14. Scale bars, 20 µm. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). One-way ANOVA with least significant difference correction (C and F), with Dunnett’s T3 correction (B, D, and E), or Kruskal-Wallis test with Dunn-Bonferroni post hoc comparison (G).
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    Image Search Results


    NCAM -1 and ALCAM are enriched in the murine VCS. (A) Scatter plots identified Ncam-1 and Alcam (red) as two of the most enriched genes with cell adhesion properties in mature PCs. (B) Quantitative RT-PCR (qPCR) confirmed significant Ncam-1 and Alcam enrichment in adult PCs in addition to other known markers of the VCS ( Cx40 and Cntn2 ) ( n =3). Error bars represent s.e.m. (C) Immunofluorescence staining in WT P21 murine hearts sections in the VCS shows overlapping expression of NCAM-1 (red) and ALCAM (red) with Cx40 (green) in the HPS. Scale bars: 25 μm.

    Journal: Development (Cambridge, England)

    Article Title: Neural cell adhesion molecule is required for ventricular conduction system development

    doi: 10.1242/dev.199431

    Figure Lengend Snippet: NCAM -1 and ALCAM are enriched in the murine VCS. (A) Scatter plots identified Ncam-1 and Alcam (red) as two of the most enriched genes with cell adhesion properties in mature PCs. (B) Quantitative RT-PCR (qPCR) confirmed significant Ncam-1 and Alcam enrichment in adult PCs in addition to other known markers of the VCS ( Cx40 and Cntn2 ) ( n =3). Error bars represent s.e.m. (C) Immunofluorescence staining in WT P21 murine hearts sections in the VCS shows overlapping expression of NCAM-1 (red) and ALCAM (red) with Cx40 (green) in the HPS. Scale bars: 25 μm.

    Article Snippet: Primary antibodies used were: NCAM-1 (1:400, rabbit, Alomone, ANR-041); PSA (1:500, rabbit, Absolute Antibody, Ab00240-23.0); SCN4B (1:1000, rabbit, Abcam, ab80539); vinculin (1:10,000, mouse Abcam, ab130007).

    Techniques: Quantitative RT-PCR, Immunofluorescence, Staining, Expressing

    Spatiotemporal expression of NCAM-1 and ALCAM during myocardial development. (A) Immunofluorescence staining of NCAM-1 (red) and Cx40 (green) during ventricular murine development at E10.5, E13.5 and E16.5. Right-hand panels show high-magnification views of the boxed areas. NCAM-1 is first detected in the compact myocardium at E13.5. By E16.5, NCAM-1 is expressed in the compact myocardium and a subset of Cx40 + cells in the trabecular myocardium. (B) Immunofluorescence staining for ALCAM (red) and Cx40 (green) during ventricular murine development at E10.5, E13.5 and E16.5. Right-hand panels show high-magnification views of the boxed areas. ALCAM is detected as early as E10.5 and its expression remains restricted to the trabecular myocardium. Scale bars: 100 μm (main panels); 25 μm (right-hand panels). (C) Immunofluorescence staining at P1 for NCAM-1 (green) and ALCAM (red) detected expression in trabecular ventricular myocardium, which becomes progressively restricted to the subendocardial region. By P7, NCAM-1 and ALCAM are restricted to PCs and absent from VMs. LV, left ventricle; RV, right ventricle. Scale bars: 25 μm.

    Journal: Development (Cambridge, England)

    Article Title: Neural cell adhesion molecule is required for ventricular conduction system development

    doi: 10.1242/dev.199431

    Figure Lengend Snippet: Spatiotemporal expression of NCAM-1 and ALCAM during myocardial development. (A) Immunofluorescence staining of NCAM-1 (red) and Cx40 (green) during ventricular murine development at E10.5, E13.5 and E16.5. Right-hand panels show high-magnification views of the boxed areas. NCAM-1 is first detected in the compact myocardium at E13.5. By E16.5, NCAM-1 is expressed in the compact myocardium and a subset of Cx40 + cells in the trabecular myocardium. (B) Immunofluorescence staining for ALCAM (red) and Cx40 (green) during ventricular murine development at E10.5, E13.5 and E16.5. Right-hand panels show high-magnification views of the boxed areas. ALCAM is detected as early as E10.5 and its expression remains restricted to the trabecular myocardium. Scale bars: 100 μm (main panels); 25 μm (right-hand panels). (C) Immunofluorescence staining at P1 for NCAM-1 (green) and ALCAM (red) detected expression in trabecular ventricular myocardium, which becomes progressively restricted to the subendocardial region. By P7, NCAM-1 and ALCAM are restricted to PCs and absent from VMs. LV, left ventricle; RV, right ventricle. Scale bars: 25 μm.

    Article Snippet: Primary antibodies used were: NCAM-1 (1:400, rabbit, Alomone, ANR-041); PSA (1:500, rabbit, Absolute Antibody, Ab00240-23.0); SCN4B (1:1000, rabbit, Abcam, ab80539); vinculin (1:10,000, mouse Abcam, ab130007).

    Techniques: Expressing, Immunofluorescence, Staining

    Ncam-1 -deficient mice display ventricular conduction slowing and a dysmorphic VCS morphology. (A) Representative surface ECG traces of adult Cntn2 , Alcam and Ncam-1 WT and KO mice. Ncam-1 KO mice show a modest but significant prolongation of the QRS interval compared with Ncam-1 WT controls. No difference in QRS was observed with Alcam - or Cntn2 -deficient mice. ( n =12 for each genotype). (B) Adult Cntn2 , Alcam and Ncam-1 KO mice backcrossed into the Cntn2 -EGFP reporter line were used to visualize the right and left HPS. Center panels are high-magnification views of the boxed areas of the left ventricle (LV). Right ventricle (RV) is displayed in the right-hand panels ( Cntn2 and Alcam KO n =5; Ncam-1 KO n =9, of which six had overt morphological defects). (C) RFP + cells dyed with TMRM were purified into ventricular (VM) and Purkinje (PC) fractions. Shown are representative FACS plots of PC (RFP + GFP + ) and VM (RFP + GFP − ) populations and the percentages of PCs relative to VMs were calculated for Ncam-1 WT and KO samples ( n =4). (D) PC population was also quantified using MATLAB in acquired GFP images from WT and Ncam-1 KO hearts. Pixel density was calculated per unit of masked region. All comparisons between WT and KO groups were made by applying a two-tailed Student's t -test ( n =4). Data represent mean±s.e.m. * P <0.05, *** P <0.001, WT versus KO. Scale bars: 1 mm.

    Journal: Development (Cambridge, England)

    Article Title: Neural cell adhesion molecule is required for ventricular conduction system development

    doi: 10.1242/dev.199431

    Figure Lengend Snippet: Ncam-1 -deficient mice display ventricular conduction slowing and a dysmorphic VCS morphology. (A) Representative surface ECG traces of adult Cntn2 , Alcam and Ncam-1 WT and KO mice. Ncam-1 KO mice show a modest but significant prolongation of the QRS interval compared with Ncam-1 WT controls. No difference in QRS was observed with Alcam - or Cntn2 -deficient mice. ( n =12 for each genotype). (B) Adult Cntn2 , Alcam and Ncam-1 KO mice backcrossed into the Cntn2 -EGFP reporter line were used to visualize the right and left HPS. Center panels are high-magnification views of the boxed areas of the left ventricle (LV). Right ventricle (RV) is displayed in the right-hand panels ( Cntn2 and Alcam KO n =5; Ncam-1 KO n =9, of which six had overt morphological defects). (C) RFP + cells dyed with TMRM were purified into ventricular (VM) and Purkinje (PC) fractions. Shown are representative FACS plots of PC (RFP + GFP + ) and VM (RFP + GFP − ) populations and the percentages of PCs relative to VMs were calculated for Ncam-1 WT and KO samples ( n =4). (D) PC population was also quantified using MATLAB in acquired GFP images from WT and Ncam-1 KO hearts. Pixel density was calculated per unit of masked region. All comparisons between WT and KO groups were made by applying a two-tailed Student's t -test ( n =4). Data represent mean±s.e.m. * P <0.05, *** P <0.001, WT versus KO. Scale bars: 1 mm.

    Article Snippet: Primary antibodies used were: NCAM-1 (1:400, rabbit, Alomone, ANR-041); PSA (1:500, rabbit, Absolute Antibody, Ab00240-23.0); SCN4B (1:1000, rabbit, Abcam, ab80539); vinculin (1:10,000, mouse Abcam, ab130007).

    Techniques: Purification, Two Tailed Test

    Dysregulation of PC programming in Ncam-1 KO mice. (A) Volcano plot of relative transcript expression from Ncam-1 WT and KO PCs ( n =3). All significantly ( P <0.05) expressed genes are labeled red (enriched) or blue (downregulated) and all nonsignificantly different transcripts are gray. (B) Functional network analysis using the DAVID functional annotation tool of differentially expressed genes from PCs of Ncam-1 WT and KO mice. (C) Table of selected targets and quantitative RT-PCR of enriched in PCs of Ncam-1 WT mice compared with PCs of Ncam-1 KO mice with log2-fold change, fold change and P -value. (D) SCN4B in PCs and VMs of Ncam-1 WT and KO mice ( n =3). Western blot analysis and quantification of adult brain (30 μg) and heart ventricular lysates (30 μg) probed for SCN4B. Vinculin was used as loading control. Data represent mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001 (two-tailed Student's t -test) WT versus KO. NS, not significant.

    Journal: Development (Cambridge, England)

    Article Title: Neural cell adhesion molecule is required for ventricular conduction system development

    doi: 10.1242/dev.199431

    Figure Lengend Snippet: Dysregulation of PC programming in Ncam-1 KO mice. (A) Volcano plot of relative transcript expression from Ncam-1 WT and KO PCs ( n =3). All significantly ( P <0.05) expressed genes are labeled red (enriched) or blue (downregulated) and all nonsignificantly different transcripts are gray. (B) Functional network analysis using the DAVID functional annotation tool of differentially expressed genes from PCs of Ncam-1 WT and KO mice. (C) Table of selected targets and quantitative RT-PCR of enriched in PCs of Ncam-1 WT mice compared with PCs of Ncam-1 KO mice with log2-fold change, fold change and P -value. (D) SCN4B in PCs and VMs of Ncam-1 WT and KO mice ( n =3). Western blot analysis and quantification of adult brain (30 μg) and heart ventricular lysates (30 μg) probed for SCN4B. Vinculin was used as loading control. Data represent mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001 (two-tailed Student's t -test) WT versus KO. NS, not significant.

    Article Snippet: Primary antibodies used were: NCAM-1 (1:400, rabbit, Alomone, ANR-041); PSA (1:500, rabbit, Absolute Antibody, Ab00240-23.0); SCN4B (1:1000, rabbit, Abcam, ab80539); vinculin (1:10,000, mouse Abcam, ab130007).

    Techniques: Expressing, Labeling, Functional Assay, Quantitative RT-PCR, Western Blot, Two Tailed Test

    PSA is expressed in the developing ventricular myocardium. (A) Western blot analysis of protein extract from whole ventricles at the specified developmental stages using anti-NCAM-1 and anti-PSA-NCAM-1 antibodies. Vinculin was used as loading control. Lysate protein amount loaded: Heart-PSA blot, 15 μg; Heart-NCAM-1 blot, 20 μg. (B) Immunofluorescence staining of NCAM-1 (red) and PSA (green) during ventricular murine development at E13.5, E14.5 and E16.5. PSA is first detected in the atria, AV junction and epicardium at E13.5. By E14.5 and for the remainder of embryonic development, PSA is also detected in NCAM-1 + trabecular cardiomyocytes in the subendocardial region. On the right, high-magnification images of the boxed area at E16.5 shows that PSA (green) and NCAM-1 (red) colocalize in the cell membrane. (C) Immunofluorescence staining of E16.5 Ncam-1 KO sequential sections shows no PSA expression in the absence of NCAM-1. (D) Tyrosine hydroxylase expression in AV junction at P3. Immunofluorescence staining of NCAM-1 (red) with TH (green) and PSA (green) at P3 showing atrioventricular junction region. Right-hand panels show high-magnification views of the boxed areas. AV, atrioventricular junction; LV, left ventricle; RV, right ventricle. Scale bars: 100 μm (B, main panels; C; D, left); 25 μm (B, high-magnification panel; D, right).

    Journal: Development (Cambridge, England)

    Article Title: Neural cell adhesion molecule is required for ventricular conduction system development

    doi: 10.1242/dev.199431

    Figure Lengend Snippet: PSA is expressed in the developing ventricular myocardium. (A) Western blot analysis of protein extract from whole ventricles at the specified developmental stages using anti-NCAM-1 and anti-PSA-NCAM-1 antibodies. Vinculin was used as loading control. Lysate protein amount loaded: Heart-PSA blot, 15 μg; Heart-NCAM-1 blot, 20 μg. (B) Immunofluorescence staining of NCAM-1 (red) and PSA (green) during ventricular murine development at E13.5, E14.5 and E16.5. PSA is first detected in the atria, AV junction and epicardium at E13.5. By E14.5 and for the remainder of embryonic development, PSA is also detected in NCAM-1 + trabecular cardiomyocytes in the subendocardial region. On the right, high-magnification images of the boxed area at E16.5 shows that PSA (green) and NCAM-1 (red) colocalize in the cell membrane. (C) Immunofluorescence staining of E16.5 Ncam-1 KO sequential sections shows no PSA expression in the absence of NCAM-1. (D) Tyrosine hydroxylase expression in AV junction at P3. Immunofluorescence staining of NCAM-1 (red) with TH (green) and PSA (green) at P3 showing atrioventricular junction region. Right-hand panels show high-magnification views of the boxed areas. AV, atrioventricular junction; LV, left ventricle; RV, right ventricle. Scale bars: 100 μm (B, main panels; C; D, left); 25 μm (B, high-magnification panel; D, right).

    Article Snippet: Primary antibodies used were: NCAM-1 (1:400, rabbit, Alomone, ANR-041); PSA (1:500, rabbit, Absolute Antibody, Ab00240-23.0); SCN4B (1:1000, rabbit, Abcam, ab80539); vinculin (1:10,000, mouse Abcam, ab130007).

    Techniques: Western Blot, Immunofluorescence, Staining, Expressing

    Generation of PSA-deficient double KO mice by CRISPR/Cas9. (A) ST8Sia2 and ST8Sia4 sgRNA (blue) targeting sequences. Stop codon is labeled with a star and protospacer adjacent motif (PAM) sequence is indicated in red. Black arrowheads indicate the site of the mutation, which is indicated in parentheses for each. Base pair sequence indicates the region where the mutation is, which is indicated with an arrow in the KO sequences by Sanger sequencing. (B) Western blot of protein extract from whole ventricles from PSA-deficient DKOs and littermate controls at P1 (10 μg). Results show that PSA is not detected in DKO mice but is still detected in Sia2 +/− /Sia4 +/− , Sia2 −/− /Sia4 +/− and Sia2 +/− /Sia4 −/− littermates. Probing with anti-NCAM-1 antibody reveals unaffected NCAM-1 levels for all heart samples. Vinculin was used as loading control. (C) Body weight and heart weight/tibia length (HR/TL) ratios of Ctrl (DHet) and PSA-deficient DKO hearts. Hematoxylin and Eosin and trichrome stain of Ctrl (DHet) and PSA-deficient DKO hearts is shown below. Data represent mean±s.e.m. *** P <0.001, **** P <0.0001 (two-tailed Student's t -test) of Ctrl (DHet) versus DKO. Scale bar: 1 mm.

    Journal: Development (Cambridge, England)

    Article Title: Neural cell adhesion molecule is required for ventricular conduction system development

    doi: 10.1242/dev.199431

    Figure Lengend Snippet: Generation of PSA-deficient double KO mice by CRISPR/Cas9. (A) ST8Sia2 and ST8Sia4 sgRNA (blue) targeting sequences. Stop codon is labeled with a star and protospacer adjacent motif (PAM) sequence is indicated in red. Black arrowheads indicate the site of the mutation, which is indicated in parentheses for each. Base pair sequence indicates the region where the mutation is, which is indicated with an arrow in the KO sequences by Sanger sequencing. (B) Western blot of protein extract from whole ventricles from PSA-deficient DKOs and littermate controls at P1 (10 μg). Results show that PSA is not detected in DKO mice but is still detected in Sia2 +/− /Sia4 +/− , Sia2 −/− /Sia4 +/− and Sia2 +/− /Sia4 −/− littermates. Probing with anti-NCAM-1 antibody reveals unaffected NCAM-1 levels for all heart samples. Vinculin was used as loading control. (C) Body weight and heart weight/tibia length (HR/TL) ratios of Ctrl (DHet) and PSA-deficient DKO hearts. Hematoxylin and Eosin and trichrome stain of Ctrl (DHet) and PSA-deficient DKO hearts is shown below. Data represent mean±s.e.m. *** P <0.001, **** P <0.0001 (two-tailed Student's t -test) of Ctrl (DHet) versus DKO. Scale bar: 1 mm.

    Article Snippet: Primary antibodies used were: NCAM-1 (1:400, rabbit, Alomone, ANR-041); PSA (1:500, rabbit, Absolute Antibody, Ab00240-23.0); SCN4B (1:1000, rabbit, Abcam, ab80539); vinculin (1:10,000, mouse Abcam, ab130007).

    Techniques: CRISPR, Labeling, Sequencing, Mutagenesis, Western Blot, Staining, Two Tailed Test

    Mislocalization of cell membrane proteins of PCs in PSA-deficient mice. (A) Immunofluorescence staining of P21 WT and PSA-deficient hearts sections with antibodies against NCAM-1 (red) and CNTN2 (green). In WT sections, PCs show colocalization of CNTN2 and NCAM-1 in the intercalated disc. PSA-deficient mice show nonoverlapping regions of CNTN2 and NCAM-1. Insets are magnified images of the dashed boxed regions. (B) Immunofluorescence staining of PCs of WT and PSA-deficient P21 hearts with antibodies against Cx40 (red) and ALCAM (green). Insets are magnified images of the dashed boxed regions showing ALCAM + PCs that are negative for Cx40. (C) Immunofluorescence staining shows incomplete localization of Cx40 or NCAM-1 (red) to the cell membrane delineated by WGA (green) in DKO PSA-deficient murine hearts at P21. Insets are magnified images of dashed boxed regions. (D) ECGs of PSA-deficient DKO ( n =10) mice and littermates with allelic combinations: DHet ( n =10), Het/KO ( n =10) and KO/Het ( n =8). PSA-deficient DKO mice displayed prolonged QRS duration compared with all three other allelic variants. Data represent mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.0001 (one-way ANOVA). (E) Left and right ventricle whole-mount dissections of DHet and PSA-deficient DKO mice in the Cntn2-EGFP background. LV, left ventricle; RV, right ventricle. (F) Representative images of 3D SEM sections of DKO and DHet P21 heart sections. Yellow arrows point to cell junctions. Color scale indicates the thickness of the three-dimensional mesh. The colors ranging from purple to yellow represent narrower to wider volumes between membranes, respectively. Scale bars: 25 μm (A-C); 1 mm (E); 1 μm (F).

    Journal: Development (Cambridge, England)

    Article Title: Neural cell adhesion molecule is required for ventricular conduction system development

    doi: 10.1242/dev.199431

    Figure Lengend Snippet: Mislocalization of cell membrane proteins of PCs in PSA-deficient mice. (A) Immunofluorescence staining of P21 WT and PSA-deficient hearts sections with antibodies against NCAM-1 (red) and CNTN2 (green). In WT sections, PCs show colocalization of CNTN2 and NCAM-1 in the intercalated disc. PSA-deficient mice show nonoverlapping regions of CNTN2 and NCAM-1. Insets are magnified images of the dashed boxed regions. (B) Immunofluorescence staining of PCs of WT and PSA-deficient P21 hearts with antibodies against Cx40 (red) and ALCAM (green). Insets are magnified images of the dashed boxed regions showing ALCAM + PCs that are negative for Cx40. (C) Immunofluorescence staining shows incomplete localization of Cx40 or NCAM-1 (red) to the cell membrane delineated by WGA (green) in DKO PSA-deficient murine hearts at P21. Insets are magnified images of dashed boxed regions. (D) ECGs of PSA-deficient DKO ( n =10) mice and littermates with allelic combinations: DHet ( n =10), Het/KO ( n =10) and KO/Het ( n =8). PSA-deficient DKO mice displayed prolonged QRS duration compared with all three other allelic variants. Data represent mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.0001 (one-way ANOVA). (E) Left and right ventricle whole-mount dissections of DHet and PSA-deficient DKO mice in the Cntn2-EGFP background. LV, left ventricle; RV, right ventricle. (F) Representative images of 3D SEM sections of DKO and DHet P21 heart sections. Yellow arrows point to cell junctions. Color scale indicates the thickness of the three-dimensional mesh. The colors ranging from purple to yellow represent narrower to wider volumes between membranes, respectively. Scale bars: 25 μm (A-C); 1 mm (E); 1 μm (F).

    Article Snippet: Primary antibodies used were: NCAM-1 (1:400, rabbit, Alomone, ANR-041); PSA (1:500, rabbit, Absolute Antibody, Ab00240-23.0); SCN4B (1:1000, rabbit, Abcam, ab80539); vinculin (1:10,000, mouse Abcam, ab130007).

    Techniques: Immunofluorescence, Staining

    (A) RT-PCR for trpm8 (236 bp) from cDNA extracted from stage 24 whole embryo or dissected spinal cord. +/− RT, in the presence or absence of the reverse transcriptase, respectively, during conversion of isolated mRNA into cDNA. (B) In situ hybridization for trpm8 in stage 24 embryos showing specific labeling in brain and spinal cord. (C) Western blot assays from egg, stage 24 wild-type whole embryo or from stage 40 whole larva, control morpholino (MO) or TRPM8-translation-blockingMO 1 (TRPM8-tbMO1) lysates. Predicted TRPM8 molecular weight [MW]: 132 kDa. Shown are representative examples of one of 3 independent experiments. β-tubulin was used as loading control. (D) Immunostained transverse section of stage 25 spinal cord (outlined). D, dorsal; V, ventral; scale bar, 20 μm; arrows indicate TRPM8 clusters in ventral neuron domains. NCAM labeling was used as counterstaining. (E) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged at 1 Hz for 90 s. Either 100 μM (−)-menthol or vehicle (0.05% DMSO)was added after 35 s of imaging and recording continued for another 60 s. Images show a menthol-responsive ventral neuron before (left, control) and after (right) addition of (−)-menthol. Colored scale shows fluorescence intensity in arbitrary units. Traces show the changes in fluorescence for the indicated cell (arrow) in both trials. (F) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged in 30-min intervals at cold (14.5°C) and warm (26.5°C) temperatures in the absence (vehicle, 0.1% DMSO) or presence of 10 μM AMTB, TRPM8 inhibitor. Scatterplots show changes in Ca 2+ spike frequency when switching temperatures in individual spinal neurons and geometric mean (black lines) from N = 3 ventral spinal cords per condition (n of neurons analyzed: DMSO, 62; AMTB, 71). Teal circles represent neurons with higher spike frequency at 14.5°C, magenta circles represent neurons with higher spike frequency at 26.5°C, and black circles represent neurons with no change in spike frequency across temperatures; ****p < 0.0001, comparison within treatments Wilcoxon matched-pairs signed rank, two-tailed test. (G) RT-PCR from cDNA collected from stage 46 larvae previously injected with 2.5 pmol standard control morpholino (Control-MO) or TRPM8-splicing-blocking morpholino (TRPM8-sbMO) shows that trpm8 mature transcript (349 bp) is not detected in TRPM8-sbMO animals. odc : ornithine decarboxylase (101 bp) as positive control. (H) TRPM8-sbMO or Control-MO containing spinal cord from stage 24 embryos were Ca 2+ -imaged for 30 min at cold temperature (14.5°C). Graph shows individual (scatterplots) and geometric mean (black lines) Ca 2+ spike frequency from N = 3 ventral spinal cords pergroup (n of neurons analyzed: Control-MO, 81; TRPM8-sbMO, 64), ****p < 0.001, Kolmogorov-Smirnov, two-tailed test. See also and .

    Journal: Current biology : CB

    Article Title: Growth at Cold Temperature Increases the Number of Motor Neurons to Optimize Locomotor Function

    doi: 10.1016/j.cub.2019.04.072

    Figure Lengend Snippet: (A) RT-PCR for trpm8 (236 bp) from cDNA extracted from stage 24 whole embryo or dissected spinal cord. +/− RT, in the presence or absence of the reverse transcriptase, respectively, during conversion of isolated mRNA into cDNA. (B) In situ hybridization for trpm8 in stage 24 embryos showing specific labeling in brain and spinal cord. (C) Western blot assays from egg, stage 24 wild-type whole embryo or from stage 40 whole larva, control morpholino (MO) or TRPM8-translation-blockingMO 1 (TRPM8-tbMO1) lysates. Predicted TRPM8 molecular weight [MW]: 132 kDa. Shown are representative examples of one of 3 independent experiments. β-tubulin was used as loading control. (D) Immunostained transverse section of stage 25 spinal cord (outlined). D, dorsal; V, ventral; scale bar, 20 μm; arrows indicate TRPM8 clusters in ventral neuron domains. NCAM labeling was used as counterstaining. (E) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged at 1 Hz for 90 s. Either 100 μM (−)-menthol or vehicle (0.05% DMSO)was added after 35 s of imaging and recording continued for another 60 s. Images show a menthol-responsive ventral neuron before (left, control) and after (right) addition of (−)-menthol. Colored scale shows fluorescence intensity in arbitrary units. Traces show the changes in fluorescence for the indicated cell (arrow) in both trials. (F) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged in 30-min intervals at cold (14.5°C) and warm (26.5°C) temperatures in the absence (vehicle, 0.1% DMSO) or presence of 10 μM AMTB, TRPM8 inhibitor. Scatterplots show changes in Ca 2+ spike frequency when switching temperatures in individual spinal neurons and geometric mean (black lines) from N = 3 ventral spinal cords per condition (n of neurons analyzed: DMSO, 62; AMTB, 71). Teal circles represent neurons with higher spike frequency at 14.5°C, magenta circles represent neurons with higher spike frequency at 26.5°C, and black circles represent neurons with no change in spike frequency across temperatures; ****p < 0.0001, comparison within treatments Wilcoxon matched-pairs signed rank, two-tailed test. (G) RT-PCR from cDNA collected from stage 46 larvae previously injected with 2.5 pmol standard control morpholino (Control-MO) or TRPM8-splicing-blocking morpholino (TRPM8-sbMO) shows that trpm8 mature transcript (349 bp) is not detected in TRPM8-sbMO animals. odc : ornithine decarboxylase (101 bp) as positive control. (H) TRPM8-sbMO or Control-MO containing spinal cord from stage 24 embryos were Ca 2+ -imaged for 30 min at cold temperature (14.5°C). Graph shows individual (scatterplots) and geometric mean (black lines) Ca 2+ spike frequency from N = 3 ventral spinal cords pergroup (n of neurons analyzed: Control-MO, 81; TRPM8-sbMO, 64), ****p < 0.001, Kolmogorov-Smirnov, two-tailed test. See also and .

    Article Snippet: Samples were permeabilized in 0.1% Triton X-100, incubated in 1% BSA blocking solution for 1 h at 4°C, stained overnight at 4°C with primary antibody for TRPM8-extracellular (1:700–1:3000, Alomone Labs), and NCAM (1:500, Developmental Studies Hybridoma Bank), and incubated for 2 h at 23°C with secondary antibody.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, In Situ Hybridization, Labeling, Western Blot, Molecular Weight, Imaging, Fluorescence, Two Tailed Test, Injection, Blocking Assay, Positive Control

    KEY RESOURCES TABLE

    Journal: Current biology : CB

    Article Title: Growth at Cold Temperature Increases the Number of Motor Neurons to Optimize Locomotor Function

    doi: 10.1016/j.cub.2019.04.072

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Samples were permeabilized in 0.1% Triton X-100, incubated in 1% BSA blocking solution for 1 h at 4°C, stained overnight at 4°C with primary antibody for TRPM8-extracellular (1:700–1:3000, Alomone Labs), and NCAM (1:500, Developmental Studies Hybridoma Bank), and incubated for 2 h at 23°C with secondary antibody.

    Techniques: Recombinant, Western Blot, In Situ, Luciferase, Reporter Assay, Isolation, Sequencing, Software

    Western Blot analysis of trkB, BDNF, Δ-FosB, Arc, synaptophysin, syntaxin-3, PSD-95 and PSA-NCAM in the Acb shell and Acb core of WT, HET and DAT KO rats after the microdialysis experiment. Histograms on the left are the densitometric analyses of the marker/GAPDH band gray optical density (O.D.) ratios. Blot lines on the right are representative samples of two animals from each experimental group. Values are means ± SEM of the values obtained by the reported number of rats per group. * P < 0.05 with respect to WT rats (one-way ANOVA followed by Tukey’s pairwise comparisons).

    Journal: Frontiers in Behavioral Neuroscience

    Article Title: Altered Sexual Behavior in Dopamine Transporter (DAT) Knockout Male Rats: A Behavioral, Neurochemical and Intracerebral Microdialysis Study

    doi: 10.3389/fnbeh.2020.00058

    Figure Lengend Snippet: Western Blot analysis of trkB, BDNF, Δ-FosB, Arc, synaptophysin, syntaxin-3, PSD-95 and PSA-NCAM in the Acb shell and Acb core of WT, HET and DAT KO rats after the microdialysis experiment. Histograms on the left are the densitometric analyses of the marker/GAPDH band gray optical density (O.D.) ratios. Blot lines on the right are representative samples of two animals from each experimental group. Values are means ± SEM of the values obtained by the reported number of rats per group. * P < 0.05 with respect to WT rats (one-way ANOVA followed by Tukey’s pairwise comparisons).

    Article Snippet: To check for antibody specificity and cross-reactivity, the anti-BDNF antibody was challenged with 200 ng of rhBDNF (Cat# B-257, Alomone Labs, Jerusalem, Israel), while the anti-PSA-NCAM antibody was preabsorbed with 500 ng of the alfa-2–8-linked sialic polymer colominic acid (Cat# sc-239576, Santa Cruz Biotechnology, USA).

    Techniques: Western Blot, Marker

    Western Blot analysis of trkB, BDNF, Δ-FosB, Arc, synaptophysin, syntaxin-3, PSD-95 and PSA-NCAM in the mPFC and VTA of WT, HET and DAT KO rats after the microdialysis experiment. Histograms on the left are the densitometric analyses of the marker/GAPDH band gray optical density (O.D.) ratios. Blot lines on the right are representative samples of two animals from each experimental group. Values are means ± SEM of the values obtained by the reported number of rats per group. * P < 0.05, ** P < 0.01 with respect to WT rats; # P < 0.05 DAT KO with respect to HET rats (one-way ANOVA followed by Tukey’s pairwise comparisons).

    Journal: Frontiers in Behavioral Neuroscience

    Article Title: Altered Sexual Behavior in Dopamine Transporter (DAT) Knockout Male Rats: A Behavioral, Neurochemical and Intracerebral Microdialysis Study

    doi: 10.3389/fnbeh.2020.00058

    Figure Lengend Snippet: Western Blot analysis of trkB, BDNF, Δ-FosB, Arc, synaptophysin, syntaxin-3, PSD-95 and PSA-NCAM in the mPFC and VTA of WT, HET and DAT KO rats after the microdialysis experiment. Histograms on the left are the densitometric analyses of the marker/GAPDH band gray optical density (O.D.) ratios. Blot lines on the right are representative samples of two animals from each experimental group. Values are means ± SEM of the values obtained by the reported number of rats per group. * P < 0.05, ** P < 0.01 with respect to WT rats; # P < 0.05 DAT KO with respect to HET rats (one-way ANOVA followed by Tukey’s pairwise comparisons).

    Article Snippet: To check for antibody specificity and cross-reactivity, the anti-BDNF antibody was challenged with 200 ng of rhBDNF (Cat# B-257, Alomone Labs, Jerusalem, Israel), while the anti-PSA-NCAM antibody was preabsorbed with 500 ng of the alfa-2–8-linked sialic polymer colominic acid (Cat# sc-239576, Santa Cruz Biotechnology, USA).

    Techniques: Western Blot, Marker

    F values and significance levels of one-way ANOVAs performed on the results shown in <xref ref-type= Figures 7 , 8 ." width="100%" height="100%">

    Journal: Frontiers in Behavioral Neuroscience

    Article Title: Altered Sexual Behavior in Dopamine Transporter (DAT) Knockout Male Rats: A Behavioral, Neurochemical and Intracerebral Microdialysis Study

    doi: 10.3389/fnbeh.2020.00058

    Figure Lengend Snippet: F values and significance levels of one-way ANOVAs performed on the results shown in Figures 7 , 8 .

    Article Snippet: To check for antibody specificity and cross-reactivity, the anti-BDNF antibody was challenged with 200 ng of rhBDNF (Cat# B-257, Alomone Labs, Jerusalem, Israel), while the anti-PSA-NCAM antibody was preabsorbed with 500 ng of the alfa-2–8-linked sialic polymer colominic acid (Cat# sc-239576, Santa Cruz Biotechnology, USA).

    Techniques: Marker

    Expression of NCAM and profilin2 in the developing cerebral cortex. (A–G) Western blot analysis of NCAM and profilin2 expression in E12, E14, E16, E18, and P0 mouse cortices. γ-Tubulin served as a control. The protein levels in E14, E16, E18, and P0 mouse cortices were quantified relative to the protein levels in E12 mouse cortices set to 1.0. n = 3 or 4 biological replicates (total NCAM and profilin2, respectively). (H) Coronal sections of control and NCAM-cKO mouse cortices were coimmunostained for NCAM and Sox2 at E12 and E14. Scale bars, 20 µm. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). One-way ANOVA with least significant difference correction (C and F), with Dunnett’s T3 correction (B, D, and E), or Kruskal-Wallis test with Dunn-Bonferroni post hoc comparison (G).

    Journal: The Journal of Cell Biology

    Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

    doi: 10.1083/jcb.201902164

    Figure Lengend Snippet: Expression of NCAM and profilin2 in the developing cerebral cortex. (A–G) Western blot analysis of NCAM and profilin2 expression in E12, E14, E16, E18, and P0 mouse cortices. γ-Tubulin served as a control. The protein levels in E14, E16, E18, and P0 mouse cortices were quantified relative to the protein levels in E12 mouse cortices set to 1.0. n = 3 or 4 biological replicates (total NCAM and profilin2, respectively). (H) Coronal sections of control and NCAM-cKO mouse cortices were coimmunostained for NCAM and Sox2 at E12 and E14. Scale bars, 20 µm. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). One-way ANOVA with least significant difference correction (C and F), with Dunnett’s T3 correction (B, D, and E), or Kruskal-Wallis test with Dunn-Bonferroni post hoc comparison (G).

    Article Snippet: Rabbit anti-NCAM (ANR-041, RRID: AB_2756690; Alomone Labs) and rabbit anti-profilin2 (ab174322, RRID: AB_2783646; Abcam) antibodies were used for immunoprecipitation and Western blot analysis.

    Techniques: Expressing, Western Blot

    Profilin2 is a novel binding partner of NCAM. (A) Coimmunoprecipitation analysis of the interaction between NCAM and profilin2 using P0 mouse brain homogenates. (B) ELISA analysis of the binding of NCAM140ICD or NCAM180ICD to immobilized profilin2. (C–E) ELISA of the binding of biotinylated NCAM140ICD-derived peptides (C), wild-type NCAM140 (aa745–753) peptide and its mutant variants with 749 LC 750 mutated to 749 AS 750 or 748 NL 749 mutated to 748 QA 749 (D), and wild-type NCAM140ICD or mutNCAM140ICD ( 749 LC 750 to 749 AS 750 mutation; E) to immobilized profilin2. n = 3 biological replicates. (F) Schematic diagram of amino acid mutations in mutNCAM140ICD. (G and H) Coronal sections of the VZ (G) and the cortex (H) of control mice were coimmunostained for profilin2, NCAM, and Sox2 (G) or Tuj1 (H). Scale bars, 50 µm. (I) Average profilin2 immunofluorescence density in each layer. (J) Percentages of profilin2 immunoreactivity in each layer. n = 9 brain slices from three mice. (K and L) Western blot analysis of levels of NCAM and profilin2 in cultured NPCs derived from E14 control and NCAM-cKO VZ/SVZ (K). The relative levels of profilin2 protein in NCAM-cKO NPCs, with the profilin2 levels in control NPCs set to 100% (L). n = 4 biological replicates. (M) Quantitative PCR analysis of the levels of profilin2 mRNA in cultured NPCs derived from E14 control and NCAM-cKO brains. Profilin2 mRNA levels in control NPCs were set to 100%. n = 5 biological replicates. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). Two-way ANOVA (B–E), one-way ANOVA with Bonferroni correction (IZ, CP, and MZ in J), Dunnett’s T3 correction (VZ/SVZ in J), Kruskal-Wallis test with Dunn-Bonferroni correction (I), and paired t test (L and M).

    Journal: The Journal of Cell Biology

    Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

    doi: 10.1083/jcb.201902164

    Figure Lengend Snippet: Profilin2 is a novel binding partner of NCAM. (A) Coimmunoprecipitation analysis of the interaction between NCAM and profilin2 using P0 mouse brain homogenates. (B) ELISA analysis of the binding of NCAM140ICD or NCAM180ICD to immobilized profilin2. (C–E) ELISA of the binding of biotinylated NCAM140ICD-derived peptides (C), wild-type NCAM140 (aa745–753) peptide and its mutant variants with 749 LC 750 mutated to 749 AS 750 or 748 NL 749 mutated to 748 QA 749 (D), and wild-type NCAM140ICD or mutNCAM140ICD ( 749 LC 750 to 749 AS 750 mutation; E) to immobilized profilin2. n = 3 biological replicates. (F) Schematic diagram of amino acid mutations in mutNCAM140ICD. (G and H) Coronal sections of the VZ (G) and the cortex (H) of control mice were coimmunostained for profilin2, NCAM, and Sox2 (G) or Tuj1 (H). Scale bars, 50 µm. (I) Average profilin2 immunofluorescence density in each layer. (J) Percentages of profilin2 immunoreactivity in each layer. n = 9 brain slices from three mice. (K and L) Western blot analysis of levels of NCAM and profilin2 in cultured NPCs derived from E14 control and NCAM-cKO VZ/SVZ (K). The relative levels of profilin2 protein in NCAM-cKO NPCs, with the profilin2 levels in control NPCs set to 100% (L). n = 4 biological replicates. (M) Quantitative PCR analysis of the levels of profilin2 mRNA in cultured NPCs derived from E14 control and NCAM-cKO brains. Profilin2 mRNA levels in control NPCs were set to 100%. n = 5 biological replicates. Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided). Two-way ANOVA (B–E), one-way ANOVA with Bonferroni correction (IZ, CP, and MZ in J), Dunnett’s T3 correction (VZ/SVZ in J), Kruskal-Wallis test with Dunn-Bonferroni correction (I), and paired t test (L and M).

    Article Snippet: Rabbit anti-NCAM (ANR-041, RRID: AB_2756690; Alomone Labs) and rabbit anti-profilin2 (ab174322, RRID: AB_2783646; Abcam) antibodies were used for immunoprecipitation and Western blot analysis.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Mutagenesis, Immunofluorescence, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction

    Profilin2 expression is downregulated specifically by profilin2 RNAi. (A) Western blot analysis of profilin2 levels in Neuro-2a cells transfected with either siProfilin2 or NC. (B) Levels of profilin2 in siProfilin2-transfected cells relative to those in NC-transfected cells, which were set to 1.0. (C and D) Quantitative PCR analysis of the levels of profilin2 (C) or profilin1 (D) mRNA in cultured NPCs transfected with either siProfilin2 (399 or 527) or NC. The mRNA levels of profilin2/1 in NC-transfected NPCs were set to 1.0. (E and F) Western blot analysis of profilin2 levels in Neuro-2a cells transfected with scrambled shRNA (GFP) or profilin2 shRNA (shProfilin2) only or cotransfected with shProfilin2 and shRNA-resistant profilin2 (Res Profilin2). The levels of profilin2 protein were quantified relative to those in GFP-transfected cells set to 1.0. (G) NCAM levels in brain homogenates loaded in different quantities (26, 53, 78, and 104 µg). Values represent mean ± SEM. n = 4 biological replicates. *, P < 0.05; **, P < 0.01 (two sided); ns, not statistically significant. Paired t test (B), one-way ANOVA with Dunnett’s T3 correction (C and D), or least significant difference correction (F).

    Journal: The Journal of Cell Biology

    Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

    doi: 10.1083/jcb.201902164

    Figure Lengend Snippet: Profilin2 expression is downregulated specifically by profilin2 RNAi. (A) Western blot analysis of profilin2 levels in Neuro-2a cells transfected with either siProfilin2 or NC. (B) Levels of profilin2 in siProfilin2-transfected cells relative to those in NC-transfected cells, which were set to 1.0. (C and D) Quantitative PCR analysis of the levels of profilin2 (C) or profilin1 (D) mRNA in cultured NPCs transfected with either siProfilin2 (399 or 527) or NC. The mRNA levels of profilin2/1 in NC-transfected NPCs were set to 1.0. (E and F) Western blot analysis of profilin2 levels in Neuro-2a cells transfected with scrambled shRNA (GFP) or profilin2 shRNA (shProfilin2) only or cotransfected with shProfilin2 and shRNA-resistant profilin2 (Res Profilin2). The levels of profilin2 protein were quantified relative to those in GFP-transfected cells set to 1.0. (G) NCAM levels in brain homogenates loaded in different quantities (26, 53, 78, and 104 µg). Values represent mean ± SEM. n = 4 biological replicates. *, P < 0.05; **, P < 0.01 (two sided); ns, not statistically significant. Paired t test (B), one-way ANOVA with Dunnett’s T3 correction (C and D), or least significant difference correction (F).

    Article Snippet: Rabbit anti-NCAM (ANR-041, RRID: AB_2756690; Alomone Labs) and rabbit anti-profilin2 (ab174322, RRID: AB_2783646; Abcam) antibodies were used for immunoprecipitation and Western blot analysis.

    Techniques: Expressing, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Cell Culture, shRNA

    NCAM enhances NPC proliferation and differentiation through profilin2. (A) Cultured NPCs transfected with siProfilin2 or NC were incubated with NCAM antibodies and BrdU. Cells were immunostained for BrdU with DAPI counterstaining. (B, E, and F) Cultured NPCs transfected with siProfilin2 or NC were incubated with NCAM antibodies or PBS and cultured in differentiation condition for 5 d. Cells were immunostained for Tuj1 (B), GFAP (E), or O4 (F) and counterstained with DAPI. (C, D, G, and H) Percentages of BrdU + DAPI + (C), Tuj1 + DAPI + (D), GFAP + DAPI + (G), and O4 + DAPI + (H) cells in the total population of DAPI + cells. (I–K) Cultured NPCs cotransfected with profilin2 shRNA (shProfilin2) and shProfilin2-resistant plasmids (Res Profilin2), shProfilin2, or control vector expressing GFP alone (GFP) were incubated with NCAM antibodies or PBS and allowed to differentiate for 3 d. Cells were immunostained for Tuj1 or GFAP. Percentages of Tuj1 + GFP + (J) or GFAP + GFP + (K) cells in the total population of GFP + cells. n = 15 microscopic fields from three biological replicates. Scale bars, 50 µm (A, F, and I) or 20 µm (B and E). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided); ns, not statistically significant. Kruskal-Wallis test with Dunn-Bonferroni post hoc correction (C) and one-way ANOVA with Bonferroni corrections (D, G, J, and K) or Dunnett’s T3 correction (H).

    Journal: The Journal of Cell Biology

    Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

    doi: 10.1083/jcb.201902164

    Figure Lengend Snippet: NCAM enhances NPC proliferation and differentiation through profilin2. (A) Cultured NPCs transfected with siProfilin2 or NC were incubated with NCAM antibodies and BrdU. Cells were immunostained for BrdU with DAPI counterstaining. (B, E, and F) Cultured NPCs transfected with siProfilin2 or NC were incubated with NCAM antibodies or PBS and cultured in differentiation condition for 5 d. Cells were immunostained for Tuj1 (B), GFAP (E), or O4 (F) and counterstained with DAPI. (C, D, G, and H) Percentages of BrdU + DAPI + (C), Tuj1 + DAPI + (D), GFAP + DAPI + (G), and O4 + DAPI + (H) cells in the total population of DAPI + cells. (I–K) Cultured NPCs cotransfected with profilin2 shRNA (shProfilin2) and shProfilin2-resistant plasmids (Res Profilin2), shProfilin2, or control vector expressing GFP alone (GFP) were incubated with NCAM antibodies or PBS and allowed to differentiate for 3 d. Cells were immunostained for Tuj1 or GFAP. Percentages of Tuj1 + GFP + (J) or GFAP + GFP + (K) cells in the total population of GFP + cells. n = 15 microscopic fields from three biological replicates. Scale bars, 50 µm (A, F, and I) or 20 µm (B and E). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided); ns, not statistically significant. Kruskal-Wallis test with Dunn-Bonferroni post hoc correction (C) and one-way ANOVA with Bonferroni corrections (D, G, J, and K) or Dunnett’s T3 correction (H).

    Article Snippet: Rabbit anti-NCAM (ANR-041, RRID: AB_2756690; Alomone Labs) and rabbit anti-profilin2 (ab174322, RRID: AB_2783646; Abcam) antibodies were used for immunoprecipitation and Western blot analysis.

    Techniques: Cell Culture, Transfection, Incubation, shRNA, Plasmid Preparation, Expressing

    NCAM enhances NPC proliferation and differentiation through profilin2-regulated actin dynamics. (A) Western blot analysis of F- and G-actin levels in cultured control and NCAM-cKO NPCs. γ-Tubulin served as a control and was enriched in the F-actin fraction containing polymerized tubulin. (B) Relative levels of G- and F-actin in NCAM-cKO NPCs. The levels of G- and F-actin in control NPCs were set to 100%. n = 4 biological replicates. (C) Cultured MEFs were cotransfected with NCAM siRNA (siNCAM) or NC and with lentiviruses coexpressing GFP and wild-type NCAM140 (NCAM) or mutant NCAM140 (mutNCAM). MEFs cotransfected with NC and lentiviruses expressing GFP only served as a control. Western blot analysis of levels of NCAM, actin, and tubulin. Lysis with the F-actin stabilization buffer solubilizes and releases NCAM to the G-actin fraction. Relative levels of NCAM protein in the G-actin fraction and the relative ratio of G- and F-actin were quantified. n = 3 biological replicates. (D) Cultured NCAM-cKO NPCs were transduced with lentiviruses coexpressing GFP and NCAM or mutNCAM. NPCs transduced with lentiviruses expressing GFP only served as a control. NPCs were stained by fluorescent phalloidin to visualize F-actin and by DNase I to visualize G-actin. (E and F) F-actin/G-actin ratios in cells are shown in D and G, respectively. n = 54 cells (E) and 21 cells (F) from three biological replicates. (G) Cultured NCAM-cKO NPCs were transduced with plasmids coencoding either GFP or profilin2 and GFP, and then they were stained with fluorescent phalloidin and DNase I. (H) Coronal VZ sections of E12 control and NCAM-cKO mice were immunostained for actin with DAPI counterstaining. White dotted lines show examples of cell boundaries. (I) The CSI for dividing cells in the VZ. n = 40 mitotic cells from three mice. (J and K) Cultured NCAM-cKO NPCs were transduced with lentiviruses coexpressing GFP and NCAM or mutNCAM, incubated with BrdU, and immunostained for BrdU with DAPI counterstaining (J). Cultured NPCs differentiated for 5–7 d were immunostained for Tuj1 and GFAP with DAPI counterstaining (K). (L–N) Percentages of BrdU + GFP + (L), Tuj1 + GFP + (M), and GFAP + GFP + (N) cells in total GFP + cell population. n = 32 microscopic fields from three biological replicates (L). n = 5 biological replicates (M and N). Scale bars, 20 µm (D, G, J, and K) or 5 µm (H). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided); ns, not statistically significant. Paired t test (B), Mann-Whitney test (I), one-way ANOVA with Dunnett’s T3 correction (C) or Bonferroni correction (M and N), and Kruskal-Wallis test with Dunn-Bonferroni post hoc comparisons (E, F, and L).

    Journal: The Journal of Cell Biology

    Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

    doi: 10.1083/jcb.201902164

    Figure Lengend Snippet: NCAM enhances NPC proliferation and differentiation through profilin2-regulated actin dynamics. (A) Western blot analysis of F- and G-actin levels in cultured control and NCAM-cKO NPCs. γ-Tubulin served as a control and was enriched in the F-actin fraction containing polymerized tubulin. (B) Relative levels of G- and F-actin in NCAM-cKO NPCs. The levels of G- and F-actin in control NPCs were set to 100%. n = 4 biological replicates. (C) Cultured MEFs were cotransfected with NCAM siRNA (siNCAM) or NC and with lentiviruses coexpressing GFP and wild-type NCAM140 (NCAM) or mutant NCAM140 (mutNCAM). MEFs cotransfected with NC and lentiviruses expressing GFP only served as a control. Western blot analysis of levels of NCAM, actin, and tubulin. Lysis with the F-actin stabilization buffer solubilizes and releases NCAM to the G-actin fraction. Relative levels of NCAM protein in the G-actin fraction and the relative ratio of G- and F-actin were quantified. n = 3 biological replicates. (D) Cultured NCAM-cKO NPCs were transduced with lentiviruses coexpressing GFP and NCAM or mutNCAM. NPCs transduced with lentiviruses expressing GFP only served as a control. NPCs were stained by fluorescent phalloidin to visualize F-actin and by DNase I to visualize G-actin. (E and F) F-actin/G-actin ratios in cells are shown in D and G, respectively. n = 54 cells (E) and 21 cells (F) from three biological replicates. (G) Cultured NCAM-cKO NPCs were transduced with plasmids coencoding either GFP or profilin2 and GFP, and then they were stained with fluorescent phalloidin and DNase I. (H) Coronal VZ sections of E12 control and NCAM-cKO mice were immunostained for actin with DAPI counterstaining. White dotted lines show examples of cell boundaries. (I) The CSI for dividing cells in the VZ. n = 40 mitotic cells from three mice. (J and K) Cultured NCAM-cKO NPCs were transduced with lentiviruses coexpressing GFP and NCAM or mutNCAM, incubated with BrdU, and immunostained for BrdU with DAPI counterstaining (J). Cultured NPCs differentiated for 5–7 d were immunostained for Tuj1 and GFAP with DAPI counterstaining (K). (L–N) Percentages of BrdU + GFP + (L), Tuj1 + GFP + (M), and GFAP + GFP + (N) cells in total GFP + cell population. n = 32 microscopic fields from three biological replicates (L). n = 5 biological replicates (M and N). Scale bars, 20 µm (D, G, J, and K) or 5 µm (H). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two sided); ns, not statistically significant. Paired t test (B), Mann-Whitney test (I), one-way ANOVA with Dunnett’s T3 correction (C) or Bonferroni correction (M and N), and Kruskal-Wallis test with Dunn-Bonferroni post hoc comparisons (E, F, and L).

    Article Snippet: Rabbit anti-NCAM (ANR-041, RRID: AB_2756690; Alomone Labs) and rabbit anti-profilin2 (ab174322, RRID: AB_2783646; Abcam) antibodies were used for immunoprecipitation and Western blot analysis.

    Techniques: Western Blot, Cell Culture, Mutagenesis, Expressing, Lysis, Transduction, Staining, Incubation, MANN-WHITNEY

    The role of NCAM in regulating the temporal generation of neurons and glia in the developing cortex. (A) NCAM expression is high in NPCs at the neurogenic period and declines at the gliogenic period. The intracellular domain of NCAM interacts with profilin2 and promotes actin polymerization in NPCs. NCAM-dependent actin regulation is required for rounding of NPCs during mitosis as well as control of NPC proliferation and temporal differentiation into cortical neurons and glia. (B) Ablation of NCAM expression in NPCs results in reduced expression of profilin2 and loss of its NCAM-dependent regulation, leading to decreased actin polymerization and reduced rounding of mitotic NPCs. This slows down cell cycle progression, reduces NPC proliferation at an early stage of neural development, delays production of cortical neurons, and leads to precocious formation of cortical glia.

    Journal: The Journal of Cell Biology

    Article Title: NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis

    doi: 10.1083/jcb.201902164

    Figure Lengend Snippet: The role of NCAM in regulating the temporal generation of neurons and glia in the developing cortex. (A) NCAM expression is high in NPCs at the neurogenic period and declines at the gliogenic period. The intracellular domain of NCAM interacts with profilin2 and promotes actin polymerization in NPCs. NCAM-dependent actin regulation is required for rounding of NPCs during mitosis as well as control of NPC proliferation and temporal differentiation into cortical neurons and glia. (B) Ablation of NCAM expression in NPCs results in reduced expression of profilin2 and loss of its NCAM-dependent regulation, leading to decreased actin polymerization and reduced rounding of mitotic NPCs. This slows down cell cycle progression, reduces NPC proliferation at an early stage of neural development, delays production of cortical neurons, and leads to precocious formation of cortical glia.

    Article Snippet: Rabbit anti-NCAM (ANR-041, RRID: AB_2756690; Alomone Labs) and rabbit anti-profilin2 (ab174322, RRID: AB_2783646; Abcam) antibodies were used for immunoprecipitation and Western blot analysis.

    Techniques: Expressing