Journal: International Journal of Molecular Sciences

Article Title: The KDET Motif in the Intracellular Domain of the Cell Adhesion Molecule L1 Interacts with Several Nuclear, Cytoplasmic, and Mitochondrial Proteins Essential for Neuronal Functions

doi: 10.3390/ijms24020932

Figure Lengend Snippet: L1 binding partners assayed in this study.

Article Snippet: The siRNAs for murine WDR5 (sc-61799), SFPQ (PSF; sc-38305), NonO (p54/nrb; sc-38164), PSPC1 (sc-152566), PPARγ (PPARgamma; sc-29456), HistHe (Histone cluster 1 H1E; sc-37975), NDUFV2 (sc-149892), Hsc70 (HSC 70; sc-35593), and SYT1 (Synaptotagmin I; sc-41311) were from Santa Cruz Biotechnology.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: The KDET Motif in the Intracellular Domain of the Cell Adhesion Molecule L1 Interacts with Several Nuclear, Cytoplasmic, and Mitochondrial Proteins Essential for Neuronal Functions

doi: 10.3390/ijms24020932

Figure Lengend Snippet: L1-55 interacts with MeCP2, HP1, and HistH1 but not with other verified or putative L1 binding partners in cultured cortical neurons. Neurons were treated with the vehicle dimethyl sulfoxide (DMSO) (+DMSO) or with the γ-secretase inhibitor DAPT and were then subjected to proximity ligation with L1 antibodies and antibodies against NDUFV2, SFPQ, NonO, PSPC1, WDR5, TOP1, hnRNP A isoforms, HistH1, Nup93, Hsc70, SYT1, impβ1, ERα, RXR, PPARγ, AR, VDR, MeCP2, or HP1γ. Nuclei are stained with DAPI (4′,6-diamidino-2-phenylindole). ( a , b ) Representative images of DMSO- and DAPT-treated neurons stained with mouse L1 antibody C-2 and a rabbit antibody against hnRNP A isoforms ( a ) or HistH1 ( b ) are shown. Scale bar: 10 µm. ( c ) The mean values + SD are from two independent experiments and show average numbers of red dots per cell after DAPT-treatment relative to control (values of vehicle control set to 100%) (**** p < 0.001; one-way ANOVA with Dunn’s multiple comparison test). Values obtained for proteins known to bind to L1-55 served as positive controls and are marked in dark gray. ( d ) Non-nuclear fractions from wild-type (WT) and L1-deficient (KO) mice were used for immunoprecipitation with mouse HistH1 or NDUFV2 antibodies immobilized to Protein G. Fractions (input) and immunoprecipitates (IP) were subjected to Western blot analysis with L1 antibody C-2. Arrows indicate L1-55 and L1-70, and the arrowhead indicates an unknown L1 band of approximately 75 kDa.

Article Snippet: The siRNAs for murine WDR5 (sc-61799), SFPQ (PSF; sc-38305), NonO (p54/nrb; sc-38164), PSPC1 (sc-152566), PPARγ (PPARgamma; sc-29456), HistHe (Histone cluster 1 H1E; sc-37975), NDUFV2 (sc-149892), Hsc70 (HSC 70; sc-35593), and SYT1 (Synaptotagmin I; sc-41311) were from Santa Cruz Biotechnology.

Techniques: Binding Assay, Cell Culture, Ligation, Staining, Control, Comparison, Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The KDET Motif in the Intracellular Domain of the Cell Adhesion Molecule L1 Interacts with Several Nuclear, Cytoplasmic, and Mitochondrial Proteins Essential for Neuronal Functions

doi: 10.3390/ijms24020932

Figure Lengend Snippet: In cultured cortical neurons, L1 interacts with several binding partners via its KDET motif. Cultured cortical neurons were treated with vehicle, tat-KDET peptide, or tat-QNQS control peptide, followed by treatment without ( a ) and with ( b ) L1 antibody 557 and proximity ligation with a L1 antibody and an antibody against MeCP2, NDUFV2, SFPQ, NonO, PSPC1, WDR5, TOP1, HistH1, Nup93, Hsc70, SYT1, ERα, or PPARγ. Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of treatment with vehicle set to 100%) (**** p < 0.001; one-way ANOVA with Bonferroni´s multiple comparison test).

Article Snippet: The siRNAs for murine WDR5 (sc-61799), SFPQ (PSF; sc-38305), NonO (p54/nrb; sc-38164), PSPC1 (sc-152566), PPARγ (PPARgamma; sc-29456), HistHe (Histone cluster 1 H1E; sc-37975), NDUFV2 (sc-149892), Hsc70 (HSC 70; sc-35593), and SYT1 (Synaptotagmin I; sc-41311) were from Santa Cruz Biotechnology.

Techniques: Cell Culture, Binding Assay, Control, Ligation, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The KDET Motif in the Intracellular Domain of the Cell Adhesion Molecule L1 Interacts with Several Nuclear, Cytoplasmic, and Mitochondrial Proteins Essential for Neuronal Functions

doi: 10.3390/ijms24020932

Figure Lengend Snippet: Reduction of SFPQ, NonO, WDR5, NDUFV2, SYT1, Hsc70, and HistH1e expression by siRNAs inhibits L1-dependent neurite outgrowth. Cortical neurons were not treated (no), treated without (mock), or treated with siRNAs specific for SFPQ, NonO, PSPC1, WDR5, NDUFV2, SYT1, Hsc70, and HistH1e. Neurons were then treated without or with antibody 557. ( a ) Mean values + SEM from three independent experiments are shown for total neurite lengths (**** p < 0.0001 relative to L1 antibody-stimulated mock-transfected neurons, #### p < 0.0001 relative to non-stimulated mock-transfected neurons; one-way ANOVA with Dunn´s multiple comparison test). ( b ) Western blot analysis of lysates from mock-transfected neurons or neurons transfected with siRNAs using the corresponding antibodies. Ponceau S staining of a prominent 35-kDa band served as loading control. ( c ) Immunostaining of mock-transfected neurons or neurons transfected with NDUFV2 siRNA using a NDUFV2 antibody. Scale bar: 10 µm.

Article Snippet: The siRNAs for murine WDR5 (sc-61799), SFPQ (PSF; sc-38305), NonO (p54/nrb; sc-38164), PSPC1 (sc-152566), PPARγ (PPARgamma; sc-29456), HistHe (Histone cluster 1 H1E; sc-37975), NDUFV2 (sc-149892), Hsc70 (HSC 70; sc-35593), and SYT1 (Synaptotagmin I; sc-41311) were from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Comparison, Western Blot, Staining, Control, Immunostaining

Journal: Molecular and Cellular Biology

Article Title: Abnormalities Caused by Carbohydrate Alterations in Iβ6- N -Acetylglucosaminyltransferase-Deficient Mice

doi: 10.1128/mcb.25.17.7828-7838.2005

Figure Lengend Snippet: FIG. 7. Estimation of synaptotagmin II and VII levels in the kidney of 6-month-old male mice by Western blot analysis. The intensity of bands determined by densitometry was normalized to that of -actin. The results are shown as a ratio to the value for wild-type mice (n 4, WT, solid bar). The value in the deficient mice (n 4, KO) was shown by an open bar.

Article Snippet: Eight-week-old, male F1 mice were used for analysis of mRNA and enzymatic assay except that in panel E F8 mice were used. on M ay 27, 2015 by LA T R O B E U N IV E R S IT Y http://m cb.asm .org/ D ow nloaded from (Santa Cruz Biotechnology), goat anti-mouse synaptotagmin II antibody S-15 (Santa Cruz Biotechnology) and goat anti-human synaptotagmin VII antibody N-18, which cross-reacts with the mouse antibody (Santa Cruz Biotechnology) or OSK-14 antibody.

Techniques: Western Blot

Journal: The FASEB Journal

Article Title: Increased levels of Aβ42 decrease the lifespan of ob/ob mice with dysregulation of microglia and astrocytes

doi: 10.1096/fj.201901028rr

Figure Lengend Snippet: FIGURE 3 Levels of neuronal markers were not altered in 18-month-old AppNL-F/wt knock-in; ob/ob mice. Levels of NF-H (A), SYT1 (B), PSD95 (C), and phospho-tau (D) were assessed using ELISAs and compared among each genotype after adjusting for sex (n = 10-44 mice/group). Standard curve of ELISAs of NF-H (E), SYT1 (F), PSD95 (G), and phospho-tau (H) with sample range as shown in red line in a representative assay. A-D, Data are presented as adjusted means ± standard errors of the means and were compared among each genotype using Tukey's HSD test. APP KI, AppNL-F/wt knock-in; APP KI ob/ob, AppNL-F/wt knock-in; ob/ob mice; NS, not significant; WT, wild type

Article Snippet: Recombinant human SYT1 proteins (Origene) were used as standards.

Techniques: Knock-In

Journal: The FASEB Journal

Article Title: Increased levels of Aβ42 decrease the lifespan of ob/ob mice with dysregulation of microglia and astrocytes

doi: 10.1096/fj.201901028rr

Figure Lengend Snippet: FIGURE 5 Levels of neuronal and glial markers in young (6-month-old) AppNL-F/wt knock-in; ob/ob mice. A, Body weight of young mice were compared among each genotype after adjusting for sex (n = 8-17 mice/group). B-F, Levels of NF-H (B), SYT1 (C), PSD95 (D), CD11b (E), and GFAP (F) in the brains of young mice were compared among each genotype after adjusting for sex (n = 9-17 mice/group). Data are presented as adjusted means ± standard errors of the means. *P < .05, **P < .01, and ***P < .001 for the comparisons among each genotype using Tukey's HSD test. APP KI, AppNL-F/wt knock-in; APP KI ob/ob, AppNL-F/wt knock-in; ob/ob mice; NS, not significant; WT, wild type

Article Snippet: Recombinant human SYT1 proteins (Origene) were used as standards.

Techniques: Knock-In

Journal: Neural Plasticity

Article Title: Brain-Specific SNAP-25 Deletion Leads to Elevated Extracellular Glutamate Level and Schizophrenia-Like Behavior in Mice

doi: 10.1155/2017/4526417

Figure Lengend Snippet: Alteration of expression pattern of SNARE-related proteins. Representative western blot (left) and densitometric analysis (right) of proteins in the cytosolic (a) and membrane (b and c) fractions prepared from mouse cerebral cortex ( n = 3 per group). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 compared with control littermates. P-SYT: phosphorylated synaptotagmin-1.

Article Snippet: Then, the lysates were separated by SDS–PAGE and probed with specific antibodies: SNAP-25 (Abcam, ab66066), SNAP-23 (Abcam, ab3340), syntaxin (Santa Cruz, sc-12736), Vamp2 (Abcam, ab6276), Munc-18 (SYSY, 116002), Phospho-Synaptotagmin (R&D Systems, PPS085), β -ACTIN (Abcam, ab6276), TUBULIN (Abcam, ab15246), and Na/K ATPase (Millipore, 05-369).

Techniques: Expressing, Western Blot, Membrane, Control

Journal: Molecular Neurobiology

Article Title: Pathological Deficit of Cystatin B Impairs Synaptic Plasticity in EPM1 Human Cerebral Organoids

doi: 10.1007/s12035-023-03812-y

Figure Lengend Snippet: Synaptic proteins in human cerebral organoids and neurons. a Western blots of the total lysate (h) and synaptosomes (syn) from hCOs using synaptophysin (SYP), sintaxin (STX), synaptotagmin 1/2 (SYT), and β-actin (ACTB) antibodies. Molecular weight in kDalton (kDa) is indicated on the left. Enrichment of b synaptophysin, c syntaxin, and d synaptotagmin 1/2 protein level, normalized on β-actin, in the synaptosomes (syn) compared to the homogenate (h, dotted line) of hCOs at 2, 3, 5, 7, 11, and 14 months (m). e Western blot of the synaptosomes from hCOs using cystatin B (CSTB) and ACTB antibodies. Molecular weight in kDa is indicated on the left. f CSTB protein level, normalized on β-actin, in synaptosomes of hCOs. m, months. Each time point is a pool of 20–40 hCOs. g Micrographs showing doublecortin (DCX, red), cystatin B (CSTB, white), and synaptophysin (SYP, green) in 10-week neurons from NPCs. DAPI (blu) stained nuclei. The arrowheads in the enlarged images of the boxed area indicate the colocalization of SYP and CSTB. Scale bar in each panel

Article Snippet: Western blot analysis was performed as previously reported [ , , ] with the following primary antibodies: SYP (1:1000, AB9272 Millipore), syntaxin (STX, 1:500, E-AB-33012 Elabscience), synaptotagmin 1/2 (SYT, 1:1000, E-AB-33005 Elabscience), CSTB (1:2000, Antikoerper AbIN271833), CD81 (1:500, AB9272 Santa Cruz), CD9 (1:500, sc-13118 Santa Cruz Biotechnology), CD82 (1:500, sc-518002 Santa Cruz Biotechnology), eIF4G2 (1:1000, HPA016965 Sigma-Aldrich), and β-actin (ACTB, 1:2000, 612,656 BD Biosciences). β-actin was used as a normalizer since its expression levels showed no substantial variations among the control and patients’ hCOs in both lysate and synaptosomal fraction (data not shown).

Techniques: Western Blot, Molecular Weight, Staining

Journal: Molecular Neurobiology

Article Title: Pathological Deficit of Cystatin B Impairs Synaptic Plasticity in EPM1 Human Cerebral Organoids

doi: 10.1007/s12035-023-03812-y

Figure Lengend Snippet: Pathological low expression levels of CSTB results in synaptic impairment in EPM1 hCOs. Western blots of a total lysate and b synaptosomal fractions from controls and EPM1 hCOs at different developmental stages using cystatin B (CSTB) and β-actin (ACTB) antibodies. Western blots of c total lysate and d synaptosomal fractions from controls and EPM1 hCOs using eukariotic Initiation Factor 4G2 (eIF4G2) and ACTB antibodies. Western blots (upper panels) and relative quantifications (lower panels). e Western blots of synaptosomes from controls and EPM1 hCOs at different developmental stages, using synaptophysin (SYP), sintaxin (STX), synaptotagmin 1/2 (SYT), and ACTB antibodies and relative quantifications. Molecular weight of each protein is indicated on the left (kDa). Data are presented as mean ± SD from n = 2 pools of 20–40 organoids. Controls: C1, C2; patients: E1, E2; m, months

Article Snippet: Western blot analysis was performed as previously reported [ , , ] with the following primary antibodies: SYP (1:1000, AB9272 Millipore), syntaxin (STX, 1:500, E-AB-33012 Elabscience), synaptotagmin 1/2 (SYT, 1:1000, E-AB-33005 Elabscience), CSTB (1:2000, Antikoerper AbIN271833), CD81 (1:500, AB9272 Santa Cruz), CD9 (1:500, sc-13118 Santa Cruz Biotechnology), CD82 (1:500, sc-518002 Santa Cruz Biotechnology), eIF4G2 (1:1000, HPA016965 Sigma-Aldrich), and β-actin (ACTB, 1:2000, 612,656 BD Biosciences). β-actin was used as a normalizer since its expression levels showed no substantial variations among the control and patients’ hCOs in both lysate and synaptosomal fraction (data not shown).

Techniques: Expressing, Western Blot, Molecular Weight

Journal: Journal of Molecular Neuroscience

Article Title: Interactions of Antibodies to the Gram-Negative Gastric Bacterium Helicobacter pylori with the Synaptic Calcium Sensor Synaptotagmin 5, Correlate to Impaired Vesicle Recycling in SiMa Human Neuroblastoma Cells

doi: 10.1007/s12031-020-01670-0

Figure Lengend Snippet: Western blot analysis of the cross-reactivity of antibodies directed to Helicobacter pylori (α- HPy ) and Campylobacter jejuni (α- CJe ) with different protein samples as provided by commercial HEK-293 overexpression lysates. a Cross-reactivity of α- HPy as revealed by a distinct immunopositive band can be observed for Syt5 and Vglut1 (Slc17a7), whereas Stmn4 and Ncan reveal no such band. Also a control lysate of non-transfected HEK293 cells, as well as an overexpression lysate of Srf is negative. b For α- CJe cross-reactivity as revealed by a distinct immunopositive band can be observed for Syt5 and Vglut1, whereas also in this case Stmn4 and Ncan reveal no such band. Again a control lysate of non-transfected HEK293 cells, as well as an overexpression lysate of Srf is negative. A corresponding negative control incubated with secondary antibodies only is shown in Supplementary Fig.

Article Snippet: Human Syt5- transfected HEK293 overexpression lysate (Origene, LY418847).

Techniques: Western Blot, Over Expression, Transfection, Negative Control, Incubation

Journal: bioRxiv

Article Title: CFTR High Expresser BEST4+ cells are pH-sensing neuropod cells: new implications for intestinal physiology and Cystic Fibrosis disease

doi: 10.1101/2025.01.24.634747

Figure Lengend Snippet: All regions are mid villus or villus tip rat epithelium. (A) CHE cells expressing BEST4 (green) have long basal pseudopodia (arrow) that extend to submucosal neurons (arrowheads) expressing TUBB3 (red). (B) A CHE cell basal pseudopod expressing BEST4 (green) extends to a submucosal neuron expressing SYT3 (yellow). (C) BEST4+ (green) CHE cells and all villus epithelial cells express NFM (red) at the basolateral membrane. (D) A MEIS1+ (magenta) CHE cell highly expresses cytoplasmic and apical S100A6 (green). (E) A CHE cell is innervated by neurons expressing CHAT (red). A goblet cell (arrow) expressing CHAT (red) is found in close proximity to the CHE cell. (F) A MEIS1+ (magenta) CHE cell highly expresses cytoplasmic and apical GC-C (green). GC-C is also in scattered in vesicular structures in villus enterocytes (G) BEST4 (green) and TUBB3 (magenta) staining shows CHEs at the same plane and close proximity to enteric neurons. Scale bars: 10 µm.

Article Snippet: TUBB3 (#NB100-1612), TUBB2B (#NBP2-46250), SYT3 (#NBP1-19320), and S100A6 (#NB110-93274SS) were purchased from Novus Biologicals.

Techniques: Expressing, Membrane, Staining

Journal: Cell reports

Article Title: Synaptotagmins 3 and 7 mediate the majority of asynchronous release from synapses in the cerebellum and hippocampus

doi: 10.1016/j.celrep.2024.114595

Figure Lengend Snippet:

Article Snippet: Mouse anti-SYT7 , Neuromab , Cat#: 75265; RRID: AB_11030371.

Techniques: Recombinant, Knock-Out, Software

Journal: The Journal of comparative neurology

Article Title: Three-Dimensional Ultrastructure of Flower-Spray Nerve Endings in the Rat Carotid Sinus.

doi: 10.1002/cne.25654

Figure Lengend Snippet: FIGURE 1 Whole mount preparation of the carotid sinus with double immunofluorescence for P2 × 3 (green) and Syt1 (red). (a) Low magnification view of the carotid sinus showing P2 × 3-immunoreactive flower-spray nerve endings. Syt1 immunoreactivity is shown in flower-spray endings and in the network of varicose nerve fibers. (b and c) Three-dimensional view of the basal surface of the terminal part of the flower-spray ending indicated in rectangle in Panel a. Arrows indicate thick parent axon for the ending. Panel b shows flower-spray endings could be distinguished from network of thin varicose nerve fibers with Syn1 immunoreactivity. (d–f) Higher magnification view of the rectangle in Panel a. (d) Punctate P2 × 3 immunoreactivity is shown in the terminal part of the endings. (e) Syt1 immunoreactivity is shown as smaller dots in the terminal parts. (f) The merged figure shows that P2 × 3 and Syt1 immunoreactivities are distinct from each other.

Article Snippet: A mouse monoclonal anti-Syt1 antibody (clone ASV48, MAB4364, R&D Systems, Minneapolis, MN, USA; RRID AB_2199304) was raised against the rat brain synaptic plasmamembrane (Matthew, Tsavaler, and Reichardt 1981).

Techniques: Immunofluorescence