synaptotagmin  (Alomone Labs)

Journal: The Journal of Neuroscience

Article Title: WT1-Expressing Interneurons Regulate Left–Right Alternation during Mammalian Locomotor Activity

doi: 10.1523/JNEUROSCI.0328-18.2018

Figure Lengend Snippet: WT1 -expressing neurons terminate in close proximity to populations of commissurally projecting interneurons. A , A 20-μm-thick section of a spinal cord 46 h after PRV-152 injection into the GS muscle on the left side and stained with antibodies to GFP (green) and WT1 (blue). At this time point, no WT1 -expressing neurons on either side of the spinal cord have taken up the tracer. Contralateral region containing WT1 -expressing cells (dashed box) is expanded to the right. B , To identify presumptive synapses on genetically defined interneuronal subtypes, we inspected the synaptotagmin + “halo” (indicated by white arrows) surrounding each labeled nuclei for WT1 + /synaptotagmin + terminals. C–F , The 20-μm-thick sections cut from a P0 WT1 CreER ROSA26 tdTomato spinal cord and stained with antibodies to tdTomato (red), the synaptic marker synatotagmin (blue), as well as (green) a nuclear marker of V2a cells ( Chx10 -, C ), V1 cells ( En1 -, D ), V0 V cells ( Evx1 -, E ), or DMRT3 -expressing dI6 cells ( F ). WT1 -expressing terminals (tdTomato + /synaptotagmin + processes) were rare or absent nearby Chx10 - or En1 -expressing cells but were commonly seen in close proximity to Evx1 + and DMRT3 + neurons. C , D , Dashed boxes are expanded to the right. E , F , Arrow in the low-magnification image indicates the specific WT1 cell of interest in the panel to the right. In magnified images, arrows indicate presumptive WT1 + axon terminals. Double labeling of these processes is confirmed in orthogonal views to the right of E , F . Scale bars: Low-magnification images, 100 μm; High-magnification images: A , C , D , 20 μm; B , E , F , 5 μm.

Article Snippet: Primary antibodies used were as follows: WT1 ), En1 (gift from Jessel laboratory, Columbia University, guinea pig, 1:1000), Chx10 ), DMRT3 ), synaptotagmin (rabbit, 1:200, Alomone Labs), Evx1 ).

Techniques: Expressing, Injection, Staining, Labeling, Marker

Journal: Yonsei Medical Journal

Article Title: Expression of Ca2+-dependent Synaptotagmin Isoforms in Mouse and Rat Parotid Acinar Cells

doi: 10.3349/ymj.2006.47.1.70

Figure Lengend Snippet: RT-PCR products encoding Ca 2+ -dependent synaptotagmins in parotid acinar cells and Western blotting of synaptotagmin 1 in exocrine glands. A, RT-PCR results of the Ca 2+ -dependent synaptotagmin family in brain microsome and parotid acinar cells from mice and rats. Lane numbers indicate synaptotagmin isoforms. B, size of PCR products encoding synaptotagmin isoforms and summary of results. Note that rat parotid acinar cells express synaptotagmin isoforms 1, 3, 4, and 7. Each result is the representative of five to eight different experiments. C, Western blotting of synaptotagmin 1 in brain, parotid, submandibular, and pancreatic acinar cells from mice and rats. The brain contains 40 µg and the parotid, submandibular, and pancreatic acinar cells contain 100 µg of proteins in each lane. Each result is the representative of four different experiments. -Ex; no extract.

Article Snippet: The membrane was then probed overnight at 4℃ with each primary antibody, anti-synaptotagmin 1 (Alomone Labs, Jerusalem, Israel) polyclonal antibody (1 : 500).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Mouse Assay, Polymerase Chain Reaction

Journal: BMC Neuroscience

Article Title: Stable silencing of SNAP-25 in PC12 cells by RNA interference

doi: 10.1186/1471-2202-7-9

Figure Lengend Snippet: Specific silencing of SNAP-25 by RNA interference . A) Immunoblots were done to assess the levels of SNAP-25, SNAP-23, synaptotagmin I, syntaxin 1A, tyrosine hydroxylase and β-actin in wild type PC12 cells, SNAP-25 knockdown cells and in control transfected cells (PC12 cells stably transfected with pG418-shRNA lacking an shRNA insert). Equal amounts of protein were loaded per lane. B) The SNAP-25 phenotype is maintained for at least 10 weeks in culture in both the parent PC12 cell line and the SNAP-25 knockdown cell line. C) Human and zebrafish SNAP-25 mRNAs are resistant to RNA interference. The specificity of the SNAP-25 shRNA was demonstrated by transiently transfecting SNAP-25 knockdown cells with plasmids designed to express SNAP-25 cDNA of rat, human, or zebrafish origin. The 19 nucleotide region of rat SNAP-25 mRNA which is targeted by the SNAP-25 shRNA differs from human SNAP-25 RNA at only 2 positions and from zebrafish SNAP-25 mRNA in 4 positions. The immunoblot shows that expression of rat SNAP-25 was silenced in the SNAP-25 knockdown cells, but human and zebrafish SNAP-25 were expressed. All three SNAP-25 cDNAs appeared to be expressed in the control transfected cells in that there was more SNAP-25 in the control transfected cells than in untransfected PC12 cells. The immunoblot was stripped and reprobed for β-actin to demonstrate approximately equal amounts of protein in each sample. D) SNAP-25 mRNA is reduced in SNAP-25 knockdown cells. RT-PCR was carried out with RNA isolated from wild type PC12 cells, SNAP-25 knockdown cells and in control transfected cells. SNAP-25 mRNA was easily detected in wild type and control transfected PC12 cells, but no SNAP-25 mRNA was detected in the SNAP-25 knockdown cells in this PCR experiments. However, if more of the reverse-transcription product was used for the PCR or if more cycles were done in the PCR reaction, some SNAP-25 mRNA was detectable in the SNAP-25 knockdown cells.

Article Snippet: ImmunoblottingLevels of SNAP-25, SNAP-23, syntaxin 1A, synaptotagmin I, tyrosine hydroxylase and β-actin in the cell lines were assessed by immunoblotting with the following antibodies: SNAP-25 (ANR-001, Alomone), SNAP-23 (DS-19; Sigma), syntaxin 1A (#573831, Calbiochem), synaptotagmin I (mAb48; Developmental Systems Hybridoma Bank, University of Iowa), tyrosine hydroxylase (#657010, Calbiochem), β-actin (JLA20; Developmental Systems Hybridoma Bank, University of Iowa), and horseradish peroxidase-labeled anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch).

Techniques: Western Blot, Transfection, Stable Transfection, shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction