synaptotagmin  (Alomone Labs)

Journal: PLoS ONE

Article Title: Disassembly of Shank and Homer Synaptic Clusters Is Driven by Soluble ?-Amyloid1-40 through Divergent NMDAR-Dependent Signalling Pathways

doi: 10.1371/journal.pone.0006011

Figure Lengend Snippet: Dispersal of synaptic mGluR1 clusters by Aβ requires NMDAR and VDCC activity. Rat cortical neurons were treated with Aβ (1 µM) for 1 h (with or without co-treatment with the NMDAR antagonist MK801, or the VDCC blocker verapamil) and then immunostained for surface mGluR1 before fixation. (A, B) Aβ downregulates surface mGluR1 (surface cluster size 66.0±2.8%, N = 10, n = 300, p

Article Snippet: Cells were incubated with anti-mGluR1 (1∶100, Alomone laboratories, Jerusalem, Israel) for 1 h at 25°C and with Alexa-488 conjugated goat anti-rabbit IgG (1∶500).

Techniques: Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: AQP3 Increases Intercellular Cohesion in NSCLC A549 Cell Spheroids through Exploratory Cell Protrusions

doi: 10.3390/ijms22084287

Figure Lengend Snippet: A549 cell aggregation is an active process. ( A ) The effect of ROCK on A549 cell aggregation. The phase-contrast micrographs show the morphologies of the A549 cells following treatment with a ROCK inhibitor, Y-27632, and the Rho activator Ⅱ. ( B ) The effects of AQP3 knockdown with siRNA on actomyosin cytoskeleton remodeling. A549 cells were stained with anti-AQP3 antibody, followed by Fluorescein-conjugated antibody (green). The actin microfilaments were stained with rhodamine-conjugated phalloidin (red), and the nuclei were stained with DAPI (blue). ( C ) The effects of AQP3 knockdown with siRNA on the apoptosis signaling pathway. Twenty-four h following siRNA transfection in 2D culture condition, the cells were further incubated in the 2D or 3D culture condition. Then, cells were harvested to confirm the effect of AQP3 knockdown on the apoptotic molecular signatures using Western blotting. α-tubulin was used as an internal control.

Article Snippet: The samples were then separated with SDS-PAGE gel and immunoblotted with the antibody against AQP3 (Alomone Labs Ltd., Jerusalem, Israel, 1/200), GAPDH (BioLegend, San Diego, CA, USA), or β-actin (Santa Cruz Biotechnology) or α-tubulin (Santa Cruz Biotechnology). β-actin, GAPDH, and α-tubulin were used as loading controls.

Techniques: Staining, Transfection, Incubation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: AQP3 Increases Intercellular Cohesion in NSCLC A549 Cell Spheroids through Exploratory Cell Protrusions

doi: 10.3390/ijms22084287

Figure Lengend Snippet: The effect of AQP3 on spatiotemporal dynamics of protrusions. ( A ) Polymerase chain reaction and ( B ) Quantitative real-time reverse transcription-polymerase chain reaction of the transcript levels of the organic hydroxyl transport genes. The data shown here represent three independent experiments, and the values represent the mean ± SEM of triplicate samples. The level of each mRNA was normalized to that of the GAPDH mRNA in the same sample and presented as the fold-change over that of the 2D culture control cells. The differences in expression levels were evaluated for significance using two-tailed t-tests with unequal variance. * p

Article Snippet: The samples were then separated with SDS-PAGE gel and immunoblotted with the antibody against AQP3 (Alomone Labs Ltd., Jerusalem, Israel, 1/200), GAPDH (BioLegend, San Diego, CA, USA), or β-actin (Santa Cruz Biotechnology) or α-tubulin (Santa Cruz Biotechnology). β-actin, GAPDH, and α-tubulin were used as loading controls.

Techniques: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Two Tailed Test

Journal: Acta Neuropathologica

Article Title: Paraneoplastic CDR2 and CDR2L antibodies affect Purkinje cell calcium homeostasis

doi: 10.1007/s00401-014-1351-6

Figure Lengend Snippet: VGCC Cav2.1 expression is up-regulated by CDR-Ab internalization. a Rat cerebellar lysates were Co-IPed with anti-Cav2.1 and then detected by anti-L7/Pcp-2 in Western blot. Cav2.1 Co-IPed with L7/Pcp-2 ( left panel ) and IPed itself (control, right panel ). Arrowheads : probed protein and nonspecific IgG* (heavy chain, 55 kDa). Left : molecular mass markers (kDa). Pathology of 125 ng/mL r CDR after 6 days correlated to VGCC. b Representative Western blot: Cav2.1 expression after r CDR treatment; c r CDR internalization induces twofold rise in Cav2.1 expression level ( n E = 19), d z-stack multiphoton micrographs: co-application of Cav2.1 antagonist ω-agatoxin (150 nM) prevents r CDR2/2L induced loss of CB + ( magenta ) and L7/Pcp-2 + ( green ) PCs; scale bars 40 μm. Stereological counting of CB + ( e ) and L7/Pcp-2 + ( f ) cells/mm 3 supported the observed neuroprotective effect of ω-agatoxin during r CDR internalization for all tested groups ( n E = 7). g Representative Western blot: Cav2.1 expression after r CDR/ω-agatoxin co-treatment; h ω-agatoxin co-treatment blocks the r CDR-induced increase in Cav2.1 expression ( n E = 7). Data are mean ± SEM. Non-parametric two-tailed paired Mann–Whitney’s U test. * p

Article Snippet: VGCC Cav2.1 is up-regulated by h CDR and r CDR antibodies To confirm the suggested interaction of L7/Pcp-2 and Cav2.1, we performed Co-IPs on rat cerebellum extract and confirmed the protein complex (Fig. a).

Techniques: Expressing, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Neurobiology

Article Title: Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development

doi: 10.1007/s12035-022-02818-2

Figure Lengend Snippet: a Representative confocal image of a nerve terminal arborization. Singly, dually, and innervated by three or more axons NMJs from YFP muscles and also images of the morphologic maturation (S1, the most inmature, and S4, almost fully differentiated, stages) of the postsynaptic clusters from P9 mice. The bar indicates 10 μm. b Confocal immunofluorescence location of α 1D L-, N-, and P/Q-type voltage-dependent calcium channels (VDCCs) at the NMJ. Triple labeling of VDCCs (green fluorescence) with syntaxin (blue fluorescence) and nAChR-α-bungarotoxin (red fluorescence) in merge images. Figure shows the presence of α 1D L-, N-, and P/Q-type-VDCC (in green) in the nerve terminal of P9 Levator auris longus (LAL) muscle endplates. The bar indicates 10 μm

Article Snippet: Primary antibodies were incubated at 4 °C overnight (rabbit anti-P/Q-type calcium channel (1:1000; ACC-001, Alomone; Jerusalem, Israel); rabbit anti-α1D L-type calcium channel (CaV1.3, 1:500; ACC-005, Alomone, Jerusalem, Israel); rabbit anti-N-type calcium channel (1:500; ACC-002, Alomone); rabbit anti-Munc18-1 (1:1000; ≠ 13414, Cell Signalling Technology; Massachusetts, USA), and rabbit anti-PKCε (1:1000; ≠ 2683, Cell Signalling Technology; Massachusetts, USA)).

Techniques: Mouse Assay, Immunofluorescence, Labeling, Fluorescence