Presence of SNAP-25 in rat mast cells

Salinas, Eva; Ventura, Javier; Córdova, L E; Quintanar, J. Luis

doi:10.1016/j.imlet.2004.05.018

Cited by 12 publications

(16 citation statements)

References 15 publications

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“…Sigma, St. Louis, MO, USA), and SNAP-25 (mouse monoclonal anti-SNAP-25. SMI-81, Sternberg Monoclonals Inc., Baltimore, MD, USA) were used at 1:100, 1:50, 1:50 and 1:25 dilutions, respectively; which are intermediate values according to manufacturer's suggestion and previous results (Salinas et al, 2004). All samples exposed to primary antibodies were incubated at 4°C overnight.…”

Section: Immunohistochemical Methodsmentioning

confidence: 99%

“…The supernatant was removed between each centrifugation, then 30 ll of sample buffer (upper buffer, 10% SDS, Glycerol, Bromophenol, and bmercaptoethanol) was added and boiled at 100°C for 5 min and later at 60°C for 15 min to obtain material to measure the rotifer sample proteins by electrophoresis. Consequently, both 30 ll of protein rotifer sample and 30 ll of protein rat brain sample as a positive control were loaded into electrophoresis gel according to Salinas et al, (2004). Therefore, SDS-PAGE (10%) electrophoresis was performed according to Laemmli buffer system (1970), the stacking gel contains upper buffer (0.5 M Trizma-base, 0.4% SDS at pH 6.8) and resolving gel contains lower buffer (1.5 M Trizma-base, 0.4% SDS at pH 8.8), these buffer solutions were added to deionized water, acrylamide, 10% ammonium persulfate, and TEMED, respectively.…”

Section: Electrophoresis and Immunoblot Methodsmentioning

confidence: 99%

“…SNAP-25 and Syntaxin-1 are located in the plasma membrane of neural, endocrine, and exocrine tissues. These proteins have been reported in glandular cells of parasitic helminthes (Quintanar & López, 2001), in frog chromaffin cells (Quintanar et al, 1998), in mast cells (Salinas et al, 2004), in the rat pituitary (Quintanar & Salinas, 2002), and in neuronal tissue of rat (Mitchell & Ryan, 2004). Nevertheless, in aquatic invertebrates there are no reports of SNAREs proteins in the literature.…”

Section: Introductionmentioning

confidence: 99%

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Identification of exocytotic membrane proteins in three rotifer species

et al. 2007

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We confirmed the presence and expression of exocytotic membrane proteins: Syntaxin-1, Syntaxin-4, SNAP-23, and SNAP-25 in the rotifers Brachionus calyciflorus, Lecane quadridentata, and Philodina roseola. These proteins were identified by immunohistochemistry and immunoblot analysis using antibodies against Syntaxin-1, Syntaxin-4, SNAP-23, and SNAP-25. The presence of these proteins were observed mainly in regions of the nervous, reproductive, and glandular systems of these rotifer species, which underlines the fundamental role of exocytotic docking and fusion membrane proteins in the vesicular release of secretory proteins. The immunoblot analysis confirmed the expression of Syntaxin-1, Syntaxin-4, and SNAP-23 in the three rotifer studied, but not of SNAP-25. This study contributes to rotifer biology by revealing the presence of conservative exocytotic machinery such as SNAREs proteins in the phylum Rotifera, including members of the Class Bdelloidea the highest taxa of metazoans where sexual reproduction does not exist.

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Section: Immunohistochemical Methodsmentioning

confidence: 99%

Section: Electrophoresis and Immunoblot Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification of exocytotic membrane proteins in three rotifer species

et al. 2007

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“…The SMI81 antibody has also been used to probe calcium-dependent release of glutamate from isolated synaptic termini (21). Finally, SMI81 has been used to detect SNAP-25 protein in non-neuronal contexts, including mast cells, neutrophils, and chromaffin cells (16,22,23).Given that SMI81 is used to follow SNAP-25 protein in brain development and pathology, we decided to precisely map the antibody epitope. Our data show that the antibody recognizes a sequence at the extreme N-terminus of SNAP-25 in brain with high efficiency, and this sequence must be present at a free N-terminal end for recognition.…”

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confidence: 99%

N-Terminal Acetylation of the Neuronal Protein SNAP-25 Is Revealed by the SMI81 Monoclonal Antibody

et al. 2009

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The monoclonal antibody SMI81 binds SNAP-25, a major player in neurotransmitter release, with high affinity and has previously been used to follow changes in the levels of this protein in neuropsychiatric disorders. We report here that the SMI81 epitope is present at the extreme N-terminus of SNAP-25 and, unusually, cannot be recognized when present as an internal sequence. Although it is known that SNAP-25 can be palmitoylated and phosphorylated in brain, we now reveal the existence of a third modification, acetylation of the N-terminus. This acetylation event greatly increases the efficiency of SMI81 antibody binding. We show that this highly specific antibody can be used for studying brain function in many vertebrate organisms.The synaptosomal-associated protein of 25 kDa 1 is a highly conserved synaptic protein and a key component of the synaptic vesicle fusion machinery (1, 2). It was initially identified as the most abundant methionine-rich protein to be rapidly transported along axons, accumulating at synaptic termini (3, 4). Levels of SNAP-25 increase during development, and SNAP-25 expression coincides with the onset of synaptogenesis and neuronal maturation (5, 6). SNAP-25 is a hydrophilic protein but is tightly associated with synaptic membranes because of the attachment of hydrophobic palmitate lipids to four clustered cysteines in the center of the molecule; indeed, SNAP-25 is the major palmitoylation target in brain (3,4,7,8).Key to our understanding of SNAP-25 protein function was the finding that it forms a stoichiometric complex with syntaxin and synaptobrevin in brain (9, 10). The central role of these three soluble NSF-attachment protein receptor (SNARE) proteins, including SNAP-25, in synaptic vesicle release was confirmed upon their identification as the targets of clostridial neurotoxin action (11). Botulinum neurotoxins A and E cleave SNAP-25 at specific points in the amino acid sequence of the C-terminus, rendering the cleaved protein unable to form the SNARE complex which in turn leads to neuromuscular paralysis and death (12, 13).In light of the importance of SNAP-25 for neurotransmission, several antibodies have been developed for recognition of this protein. One such commonly used antibody is mouse monoclonal SMI81, initially generated by Sternberger Monoclonals among a wide panel of antibodies raised against human brain extract (hence the abbreviation SMI81 for Sternberger Monoclonals Inc., clone 81). The SMI81 antibody was later shown to specifically recognize a single 25 kDa protein band by Western immunoblotting in wild-type neurons, but not in neurons from a SNAP-25 null mutant background (14). It recognizes botulinum toxin-truncated protein (15-17) and both alternatively spliced SNAP-25A and -B isoforms, which differ by only nine amino acids. It has been used to detect SNAP-25 in hippocampal slices from mice at various stages of embryonic and postnatal development (18). SMI81 immunoblotting of brain regions of mice suffering from hyperkinesis showed changes in the SNA...

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“…Primary antibodies LH (AntiRat LH serum for RiA), PRL (Anti-Rat PRL serum for RiA), FSH (Anti-Rat FSH serum for RiA) and TSH (Anti-Rat TSH serum for RiA) were used at 1:20, 1:30, 1:50 and 1:100 dilutions, respectively; which are intermediate values according to manufacturer's suggestion and previous results (Salinas et al 2004). All samples exposed to primary antibodies were incubated at 4°C overnight.…”

Section: Rotifer Collection and Culturementioning

confidence: 99%

Immunodetection of Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH) and Prolactin (PRL) in Brachionus calyciflorus (Rotifera: Monogononta)

Alvarado-Flores¹,

Montoya-Garcia²,

Ventura-Juárez³

et al. 2008

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Abstract:The endocrine system controls and coordinates behavioral, biochemical, and physiological processes through signal mechanisms using neuropeptides or products of neurosecretory cells. Among invertebrates, this system is poorly studied in rotifers, in which estrogens and androgens significantly affect sexual reproduction. This is the first report of the presence of the Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH) and Prolactin (PRL) in rotifers. Analyses included the avidin-biotin-peroxidase complex method with primary antibodies LH (Anti-Rat LH serum for RiA), PRL (Anti-Rat PRL serum for RiA), FSH (Anti-Rat FSH serum for RiA) and TSH (Anti-Rat TSH serum for RiA). These hormones were found in females, males and parthenogenetic and sexual eggs of the freshwater Brachionus calyciflorus. The immunoreactivity of FSH, LH, TSH and PRL in females was observed in: ovaries, cerebrum, mastax, stomach, lorica, and the stomach gland. However, in males LH was observed only at the trochal disk and cerebrum. The hormones FSH, TSH and PRL, were observed in testicles, contractil vesicles, and cementary gland of males. Regarding amictic or parthenogenetic eggs, the hormones LH, FSH, TSH, and PRL were located mainly in the micromeres, and the staining in the macromeres was weak. On the other hand, in the mictic or sexual eggs the inner shell is stained for the hormones PRL and LH, opposite to the staining of FSH and TSH, located mainly in the embryo. in general, immuno-reactivity was observed in areas important for the reproductive, excretory, digestive and developmental processes. Rev. Biol. Trop. 57 (4):

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Presence of SNAP-25 in rat mast cells

Cited by 12 publications

References 15 publications

Identification of exocytotic membrane proteins in three rotifer species

Identification of exocytotic membrane proteins in three rotifer species

N-Terminal Acetylation of the Neuronal Protein SNAP-25 Is Revealed by the SMI81 Monoclonal Antibody

Immunodetection of Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH) and Prolactin (PRL) in Brachionus calyciflorus (Rotifera: Monogononta)

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