Detection of G Proteins in Bovine Brain Clathrin Coated Vesicles with Common Alpha and Beta Subunits Antibodies.

Silva, Walter I.

doi:10.1254/jjp.56.97

Cited by 3 publications

(2 citation statements)

References 13 publications

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“…SDS-PAGE and immunoblotting analysis of the pellets and cytosolic fractions from above was done as previously described. 13,24,27 In brief, equal total protein amounts (cytosolic and particulate fractions) or volumes (gradient fractions), plus the molecular weight standards were loaded in a 1 mm thick 12% SDS polyacrylamide gel and electrophoresed for 45 min at a constant 200 V in a Mini Protean II (Bio-Rad, Melville, NY). After electrophoresis the gel was equilibrated in transfer buer (25 mM Tris, 190 mM Glycine, 20% MeOH, 0.1% SDS) for 30 min, and then transferred overnight to a PVDF membrane using a Mini Trans-Blot apparatus (Bio-Rad, Melville, NY) at 48C and a constant 25 V. The PVDF membranes were stained with India ink to verify transfer eciency.…”

Section: Sds-page and Immunoblottingmentioning

confidence: 99%

Identification of caveolae and caveolin in C6 glioma cells

Silva

Maldonado

Lisanti

et al. 1999

Intl J of Devlp Neuroscience

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Caveolae (CAV) constitute a novel subcellular transport vesicle that has received special attention based on its proven and postulated participation in transcytosis, potocytosis, and in cell signaling events. One of the principal components of CAV are caveolin protein isoforms. Here, we have undertaken the immunochemical identification of CAV and the known caveolin isoforms (1alpha, 1beta, 2 and 3) in cultured rat C6 glioma cells. Immunoblot analysis revealed that particulate fractions from rat C6 glioma cells express caveolin-1 and caveolin-2. The relative detergent-insolubility of these caveolin isoforms was also determined by Western blot analysis. Indirect immunofluorescence analysis with caveolin-1 and -2 antibodies revealed staining patterns typical of CAV's known subcellular distribution and localization. For both caveolin isoforms immunocytochemical staining was characterized by intensely fluorescent puncta throughout the cytoplasm and diffuse micropatches at the level of the plasmalemma. Perinuclear staining was also detected, consistent and suggestive of caveolin's localization in the trans Golgi region. The caveolin-1 and -2 immunoreactivity seen in Western blots and immunocytochemically is related to structurally relevant CAV as supported by the isolation of caveolin-enriched membrane complexes using two different methods. Light-density, Triton X-100-insoluble caveolin-1- and caveolin-2-enriched fractions were obtained after fractionation of rat C6 glioma cells and their separation over 5-40% discontinuous sucrose-density gradients. Similar fractions were obtained using a detergent-free, sodium carbonate-based fractionation method. These results further support the localization of CAV and caveolins in glial cells. In addition, they demonstrate that cultured C6 glioma cells can be useful as a model system to study the role of CAV and caveolins in subcellular transport and signal transduction events in glial cells and the brain.

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Section: Sds-page and Immunoblottingmentioning

confidence: 99%

Identification of caveolae and caveolin in C6 glioma cells

Silva

Maldonado

Lisanti

et al. 1999

Intl J of Devlp Neuroscience

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“…Some of these receptors are coupled with effector systems in bovine brain CVs, i.e ., adenosine A, receptors coupled with G; protein to adenylate cyclase; metabotropic glutamate receptors coupled with phospholipase C via G protein; muscarinic acetylcholine receptors coupled with G protein (Silva et al ., 1986;González-Calero et al ., 1990, 1992Ros et al ., 1990;Martín et al ., 1991 b). In addition, high-affinity binding sites for [3H]GTP and both G; (M~39,000-40,000) and GS (M~45,000 and 52,000) proteins were found in CVs from bovine cerebral cortex (Moroi et al ., 1991 ;Silva, 1991) .…”

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Dopamine D₁ and D₂ Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue

Ozaki

Moroi

Kadota

et al. 1994

Journal of Neurochemistry

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Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D, and D Z receptors, G proteins, and adenylate cyclase . Dopamine receptors in CVs were characterized by a dopamine D, receptor antagonist, [3 H]SCH 23390, and a dopamine D2 receptor antagonist, [ 3 H]spiroperidol . The bindings of both ligands were specifically saturable and reversible with a dissociation constant (K p) of 0 .65 and 0 .5 nM, respectively . Dopaminergic antagonists and agonists inhibited the specific bindings of [ 3H]SCH 23390 and [3 H]spiroperidol in a stereoselective and concentration-dependent manner with an appropriate rank order potency for dopamine D, or D 2 receptors . The regulations of the agonist binding by guanyl-5-ylimidodiphosphate were observed . ADP ribosylation of the CVs with [32 P]NAD demonstrated predominant labeling of bands of M, 47,000-52,000, 42,000-45,000, and 40,000-39,000, which corresponded to the known molecular weights of the a subunits of GS and G ; proteins . The presence of a and ß subunits of G proteins in the CVs was also confirmed by immunoblotting assay . Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D Z receptor agonists, was present in the CVs . These findings suggest that the dopamine D,

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G-proteins in rat blood vessels—I. identification

Abebe

Edwards

Agrawal

1995

General Pharmacology: The Vascular System

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Detection of G Proteins in Bovine Brain Clathrin Coated Vesicles with Common Alpha and Beta Subunits Antibodies.

Cited by 3 publications

References 13 publications

Identification of caveolae and caveolin in C6 glioma cells

Identification of caveolae and caveolin in C6 glioma cells

Dopamine D₁ and D₂ Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue

G-proteins in rat blood vessels—I. identification

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Detection of G Proteins in Bovine Brain Clathrin Coated Vesicles with Common Alpha and Beta Subunits Antibodies.

Cited by 3 publications

References 13 publications

Identification of caveolae and caveolin in C6 glioma cells

Identification of caveolae and caveolin in C6 glioma cells

Dopamine D1 and D2 Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue

G-proteins in rat blood vessels—I. identification

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Product

Resources

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Dopamine D₁ and D₂ Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue