polyclonal anti human ncam antibody  (Proteintech)

Journal: Frontiers in Genetics

Article Title: Haploinsufficiency of the Attention-Deficit/Hyperactivity Disorder Risk Gene St3gal3 in Mice Causes Alterations in Cognition and Expression of Genes Involved in Myelination and Sialylation

doi: 10.3389/fgene.2021.688488

Figure Lengend Snippet: Expression analysis of genes involved in the polysialylation process. (A) Relative mRNA level of genes involved in the (poly)sialylation process in the hippocampus of St3gal3 mice (HET: n = 21, WT: n = 13). A significant genotype effect on Cadm1 levels was observed (genotype: F 1 , 43 = 4.124, p = 0.0485). We detected a genotype main effect (genotype: F 1 , 43 = 5.624, p = 0.0223) as well as a significant genotype × sex interaction ( F 1 , 43 = 4.736, p = 0.0351) on the levels of St8sia2 expression; post hoc analysis revealed an upregulation in HET male mice compared to WT male mice ( post hoc p = 0.0083) and HET females ( post hoc p = 0.0481). A genotype effect was also observed for St8sia4 (genotype: F 1 , 43 = 7.538, p = 0.0088). (B) Protein levels of the adhesion molecules NCAM (isoforms NCAM-180, NCAM-140, and NCAM-120) and SynCAM 1 in the hippocampus of St3gal3 male mice (HET: n = 7, WT: n = 8). Representative blots are shown in each case. No differences were observed for any of the analyzed proteins. (C) Relative mRNA level of genes involved in the polysialylation process in the PFC of St3gal3 mice (HET: n = 19, WT: n = 14). A significant sex effect was observed in Cadm1 (sex: F 1 , 42 = 5.495, p = 0.0239). Trends toward sex and genotype effects were found for Ncam1 (sex: F 1 , 42 = 3.105, p = 0.0853; genotype: F 1 , 42 = 2.981, p = 0.0916). A significant sex effect on St8sia4 expression was also detected ( F 1 , 42 = 5.558, p = 0.0231). (D) Protein levels of the adhesion molecules NCAM (isoforms NCAM-180, NCAM-140, and NCAM-120) and SynCAM 1 in the hippocampus of St3gal3 male mice (HET: n = 7, WT: n = 8). Representative blots are shown in each case. No differences were observed for any of the analyzed proteins. qPCR data were analyzed by two-way ANOVA followed by Sidak post hoc test (when necessary) and presented as mean ± SEM. Western blot data were analyzed by Student’s t -test with Welch’s correction and presented as violin plots with median + quartiles. # p < 0.1, * p < 0.05, ** p < 0.01. For simplicity, only genotype effects are indicated in the figures.

Article Snippet: A total of 20 μg of protein were loaded on 4–12% Bis-Tris protein gels (Thermo Fisher Scientific, Dreieich, Germany), electroblotted to a 0.45 μm pore nitrocellulose membrane (Thermo Fisher Scientific, Dreieich, Germany) and probed using one of the following primary antibodies, always in combination with either rabbit anti-α-tubulin antibody or goat anti- beta-tubulin (Abcam, Cambridge, United Kingdom) as loading controls: goat polyclonal anti-NCAM1/CD56 antibody (1:2,000, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-PSA antibody (1:500, #NBP2-52710, Novus, Lingen, Germany), rabbit polyclonal anti-SynCAM1/2/3 (1:2000, Synaptic Systems, Göttingen, Germany), rabbit polyclonal anti-Myelin PLP (1:5,000, #ab28486, Abcam, Cambridge, United Kingdom), rabbit polyclonal anti-MBP (1:1,000, #PD004, MBL, Woburn, MA, United Kingdom).

Techniques: Expressing, Western Blot

Journal: Nature Communications

Article Title: Internalization of a polysialic acid-binding Escherichia coli bacteriophage into eukaryotic neuroblastoma cells

doi: 10.1038/s41467-017-02057-3

Figure Lengend Snippet: Bacteriophage PK1A2 binds specifically to mammalian polysialic acid-expressing cells. a Fluorescence microscopic images of cells incubated with FITC-labeled PK1A2 phages (green) and neural cell adhesion molecule NCAM antibodies (red) to assess phage surface binding. Polysialic acid-containing (polySia+) human neuroblastoma SK-N-SH, kSK-N-SH and SH-SY5Y cells as well as human polysialic acid-negative (polySia−) neuroblastoma SK-N-AS and fibroblast BJ cells were incubated with FITC-labeled phages for 1 h at room temperature and stained for NCAM, the carrier protein of polysialic acid. b Inhibition of phage binding to kSK-N-SH cells by pretreatment with endosialidase or incubation in the presence of free polysialic acid. As a control, phage containing catalytically active endosialidase as the binding agent was used. Nuclei were stained with DAPI (blue). Representative images from two to three biological replicates are shown. The scale bars represent 20 µm

Article Snippet: For the staining of the protein carrier of polysialic acid, NCAM, rabbit polyclonal anti-human NCAM antibody (AB5032, Millipore) diluted 1:250 in PBS was used, followed by Alexa Fluor 555 goat anti-rabbit secondary antibody (A-21429, Molecular Probes) diluted 1:500 in PBS, each for 2 h at room temperature.

Techniques: Expressing, Fluorescence, Incubation, Labeling, Binding Assay, Staining, Inhibition, Control