A general radiochemical—Color method for quantitation of immunoblots

Esmaeli-Azad, Babak; Feinstein, Stuart C.

doi:10.1016/0003-2697(91)90101-x

Cited by 24 publications

(25 citation statements)

References 13 publications

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“…The development of the processes by the transfected cells probably was due to increased stabilization of microtubules produced by a combination of the expressed tau and the endogenous MAPS. These findings are consistent with those of Esmaeli-Azad, et al [13] who showed that the neurite outgrowth was directly proportional to the levels of tau expression in PC 12 cells transfected with tau.…”

Section: Discussionsupporting

confidence: 93%

“…buffered saline: 200 mM NaC1, 10 mM Na2HPO 4, pH 7.0) at 37°C and fixed for 5 min at 37°C with 4% paraformaldehyde and 0.1% Triton X-100 in 100 mM PIPES, 5 mM EGTA, 2 mM MgC12. In some cases cells were first extracted with 0.1% Triton X-100 for 1-2 min at 37°C after incubating in a microtubule-stabilizing buffer (100 mM PIPES, 5 mM EGTA, 2 mM MgCI:, 2 M glycerol, pH 6.8) [12,13]. After fixation, the cells were overlaid with ~ -20°C cold methanol for five minutes.…”

Section: Cell Culture and Geneticin G418 Selectionmentioning

confidence: 99%

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Phosphorylation and accumulation of tau without any concomitant increase in tubulin levels in Chinese Hamster Ovary cells stably transfected with human tau₄₄₁

Haque¹,

Denman²,

Merz³

et al. 1995

FEBS Letters

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Eucaryotic expression vectors bearing a 1.4 kb eDNA encoding the 4 repeat isoform of human tau, tau441, in either the sense or anti-sense orientation with respect to a cytomegalovirus (CMV) promoter were constructed. The resulting constructs were used to transiently express tau in Chinese Hamster Ovary cells and to generate non-neuronal stable cell lines. Immunocytochemical studies of these cells show that tau is expressed in the sense but not the anti-sense or vector containing lines. Some of the cells expressing tau showed fine elongated processes which were stained by tau antibodies. The general tau immunostaining pattern appeared diffuse and punctuate. The expressed tau was seen both unbound and bound to microtubules. In some cells labeling with antibodies that specifically recognize hyperphosphorylation of tau was observed. The size of this population increased with increasing numbers of cell passages. However, no increase in steady-state tubulin level was observed following tau44~ expression. These studies show that tau can accumulate in the cells without a concomitant increase in tubulin.

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Section: Discussionsupporting

confidence: 93%

Section: Cell Culture and Geneticin G418 Selectionmentioning

confidence: 99%

Phosphorylation and accumulation of tau without any concomitant increase in tubulin levels in Chinese Hamster Ovary cells stably transfected with human tau₄₄₁

Haque¹,

Denman²,

Merz³

et al. 1995

FEBS Letters

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“…Tau is also involved in the stabilization of microtubules and consequently in neurite morphology (17). Tau antisense oligonucleotides decrease neurite extension of PC12 cells, whereas Tau overexpression renders neurites resistant to nocodazole (20). Attenuation of MAP1B expression by antisense oligonucleotides also inhibits initiation of neurite outgrowth (21).…”

Section: Discussionmentioning

confidence: 99%

Microtubule-associated protein 2 (MAP2) is a neurosteroid receptor

Fontaine-Lenoir

Chambraud

Fellous

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

111

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The neurosteroid pregnenolone (PREG) and its chemically synthesized analog 3β-methoxypregnenolone (MePREG) bind to microtubule-associated protein 2 (MAP2) and stimulate the polymerization of microtubules. PREG, MePREG, and progesterone (PROG; the physiological immediate metabolite of PREG) significantly enhance neurite outgrowth of nerve growth factor-pretreated PC12 cells. However, PROG, although it binds to MAP2, does not increase the immunostaining of MAP2, contrary to PREG and MePREG. Nocodazole, a microtubule-disrupting agent, induces a major retraction of neurites in control cultures, but pretreatment with PREG/MePREG is protective. Decreasing MAP2 expression by RNA interference does not modify PROG action, but it prevents the stimulatory effects of PREG and MePREG on neurite extension, showing that MAP2 is their specific receptor.

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“…Tau is a natively unstructured protein that is localized primarily to neuronal cell bodies and axons (15,22). Tau expression is important for the establishment of normal axonal morphology and function (23)(24)(25)(26). Furthermore, pathological tau function is implicated in the etiology of neurodegenerative diseases, such as Alzheimer's disease, Pick's disease, and frontotemporal dementia with Parkinsonism linked to chromosome 17 (27).…”

mentioning

confidence: 99%

Tau induces cooperative Taxol binding to microtubules

Ross

Santangelo

Makrides

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Taxol and tau are two ligands that stabilize the microtubule (MT) lattice. Taxol is an anti-mitotic drug that binds ␤ tubulin in the MT interior. Tau is a MT-associated protein that binds both ␣ and ␤ tubulin on the MT exterior. Both Taxol and tau reduce MT dynamics and promote tubulin polymerization. Tau alone also acts to bundle, stiffen, and space MTs. A structural study recently suggested that Taxol and tau may interact by binding to the same site. Using fluorescence recovery after photobleaching, we find that tau induces Taxol to bind MTs cooperatively depending on the tau concentration. We develop a model that correctly fits the data in the absence of tau, yields the equilibrium dissociation constant of Ϸ2 M, and determines the escape rate of Taxol through one pore to be 1.7 ؋ 10 3 (M⅐s) ؊1 . Extension of the model yields a measure of Taxol cooperativity with a Hill coefficient of at least 15 when tau is present at a 1:1 molar ratio with tubulin.B ecause of their essential role in cell division, microtubules (MTs) are a major target for antimitotic cancer therapeutics such as paclitaxel (Taxol) (for reviews, see refs. 1 and 2). Taxol stabilizes the intrinsically labile MT polymer, promotes MT assembly, and suppresses MT dynamics (3-5), possibly by strengthening lateral contacts between tubulin dimers (6, 7). In addition, Taxol affects MT structure by decreasing protofilament number (8) and increasing MT flexibility (9, 10). The Taxolbinding site has been localized to the luminal face of ␤ tubulin and is accessible through 2-nm pores in the MT wall (11-13).In postmitotic differentiated neuronal cells, MTs form a major component of axons and dendrites and are thus essential for nervous system development and function. In this context, MT dynamics and functions are regulated by MT-associated proteins (MAPs) such as tau (14). In vitro, as well as in vivo, tau stabilizes and promotes MT assembly, as well as stiffens, bundles, and spaces MTs (9, 14-21). Tau is a natively unstructured protein that is localized primarily to neuronal cell bodies and axons (15,22). Tau expression is important for the establishment of normal axonal morphology and function (23-26). Furthermore, pathological tau function is implicated in the etiology of neurodegenerative diseases, such as Alzheimer's disease, Pick's disease, and frontotemporal dementia with Parkinsonism linked to chromosome 17 (27).In the central nervous system, as a result of alternative splicing of two N-terminal exons and one C-terminal exon, tau is expressed as six major isoforms. Structurally, tau consists of two major ''domains.'' The N terminus, or ''projection domain,'' is involved in bundling and spacing MTs (18,19). The C terminus, the ''MTbinding domain,'' is composed of either three or four repeated motifs. This region binds to the exterior of preassembled MTs (28-31).A recent, intriguing cryoelectron microscopy study found that the MT-binding region can bind inside the MT lumen near the Taxolbinding site (32). Before this report, tau had been thought to bi...

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A general radiochemical—Color method for quantitation of immunoblots

Cited by 24 publications

References 13 publications

Phosphorylation and accumulation of tau without any concomitant increase in tubulin levels in Chinese Hamster Ovary cells stably transfected with human tau₄₄₁

Phosphorylation and accumulation of tau without any concomitant increase in tubulin levels in Chinese Hamster Ovary cells stably transfected with human tau₄₄₁

Microtubule-associated protein 2 (MAP2) is a neurosteroid receptor

Tau induces cooperative Taxol binding to microtubules

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A general radiochemical—Color method for quantitation of immunoblots

Cited by 24 publications

References 13 publications

Phosphorylation and accumulation of tau without any concomitant increase in tubulin levels in Chinese Hamster Ovary cells stably transfected with human tau441

Phosphorylation and accumulation of tau without any concomitant increase in tubulin levels in Chinese Hamster Ovary cells stably transfected with human tau441

Microtubule-associated protein 2 (MAP2) is a neurosteroid receptor

Tau induces cooperative Taxol binding to microtubules

Contact Info

Product

Resources

About

Phosphorylation and accumulation of tau without any concomitant increase in tubulin levels in Chinese Hamster Ovary cells stably transfected with human tau₄₄₁

Phosphorylation and accumulation of tau without any concomitant increase in tubulin levels in Chinese Hamster Ovary cells stably transfected with human tau₄₄₁