The characterization and use of different antibodies against the hsp70 major heat shock protein family for the development of an immunoassay

Margulis, Boris A.; Nacharov, P. V.; Tsvetkova, Olga I.; Welsh, Michael; Kinev, Alexander

doi:10.1002/elps.1150120913

Cited by 11 publications

(2 citation statements)

References 18 publications

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“…Equal amounts (20 ,ug) of protein were then run on 12% SDS-polyacrylamide gels. After electrophoresis, proteins were electrically transferred to nitrocellulose filters and incubated with rabbit anti-rat HO antibody (StressGen Biotechnologies, Victoria, British Columbia, Canada) or rabbit anti-bovine hsp7o antibody (19) diluted 1:1000 in PBS + 5 % fat-free milkpowder. Horseraddish peroxidase (HRP) linked goat anti rabbit Ig was used as a second layer.…”

Section: Methodsmentioning

confidence: 99%

“…Binding of anti-hsp70 antibodies to mouse, rat, and human hsp7o in immunoblot experiments Hsp7O was purified from human U937 cells (Lanes A and B), mouse NIH3T3 cells (Lanes C and D), and rat RINm-5F cells (Lanes E and F). Two (Lanes A, C, and E) and 10 ng (Lanes B, D, and F) of the purified hsp7o were run on a SDS-polyacrylamide gel, blotted onto nitrocellulose, and incubated with anti-hsp7o antibodies (19). Binding was quantified using ECL and fluorography.…”

Section: Dna Nick End-labeling Of Tissue Sections (Tunel Staining)mentioning

confidence: 99%

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Differences in the Expression of Heat-Shock Proteins and Antioxidant Enzymes between Human and Rodent Pancreatic Islets: Implications for the Pathogenesis of Insulin-Dependent Diabetes Mellitus

Welsh

Margulis

Borg

et al. 1995

Mol Med

187

121

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Background: It has previously been observed that the insulin-producing cells of human pancreatic islets are more resistant to alloxan-, streptozotocin-, nitroprusside-, or cytokine-induced injury than those of mouse and rat islets. Materials and Methods: Human pancreatic islets were obtained from heart-beating organ donors. The expression of the stress proteins heat shock protein 70 (hsp7o) and heme oxygenase and the anti-apoptosis gene bcl-2 was determined in isolated rat, mouse, and human islets, either cultured in vitro or transplanted under the kidney capsule of nude mice, using immunoblot analysis. Rat and human islet sensitivity to hydrogen peroxide was assessed by glucose oxidation measurements. Isolated islets were also analyzed for their catalase and superoxide dismutase activities, and the islet cell levels of reduced glutathione were determined in response to hydrogen peroxide and nitroprusside. Programmed cell death in human and rat islets in response to streptozotocin was evaluated using TUNEL staining. Results: Cultured human islets expressed higher contents of hsp7O than mouse and rat islets at basal conditions. Also after 4 weeks under the kidney capsule of normoglycemic mice, the hsp7o levels were higher in human islets than in rat islets. The expression of another stress protein, heme oxygenase (HO), was strongly increased in cultured rat islets, but was not affected in human islets. Expression of the bcl-2 gene could not be detected in human islets. In spite of this, 0.5 mM streptozotocin induced apotosis in rat but not in human islet cells. Hydrogen peroxide (0.1 and 0.4 mM) decreased glucose oxidation rates in rat but not in human islets. The levels of reduced glutathione were moderately decreased in human and rat islet cells and sharply decreased in mouse islet cells in response to hydrogen peroxide. Moreover, the activities of catalase and superoxide dismutase (SOD) were markedly lower in mouse islets than in human islets. The activity of catalase was lower in rat islets than in human islets. Conclusion: Human islets differ clearly from mouse and rat islets in their increased expression of hsp7o, catalase, and SOD, which may explain the increased resistance of human islets to 3 cell toxins.

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Section: Methodsmentioning

confidence: 99%

Section: Dna Nick End-labeling Of Tissue Sections (Tunel Staining)mentioning

confidence: 99%

Differences in the Expression of Heat-Shock Proteins and Antioxidant Enzymes between Human and Rodent Pancreatic Islets: Implications for the Pathogenesis of Insulin-Dependent Diabetes Mellitus

Welsh

Margulis

Borg

et al. 1995

Mol Med

187

121

View full text Add to dashboard Cite

show abstract

Specific cross‐reactivity of antibodies raised against two major stress proteins, stress 70 and chaperonin 60, in diverse species

Sanders

Martin

Nakagawa

et al. 1994

Enviro Toxic and Chemistry

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Immunoblot analysis using several antibodies raised against two major families of stress proteins, stress 70 and chaperomn 60 (cpn60), which are highly conserved in mammals, was carried out in diverse species often used in environmental research, including molluscs, annelids, crustaceans, echinoderms, and fish The study revealed surprisingly different patterns of antibody cross reactivity among species The monoclonal anti‐stress 70 antibody (mAb) C92 was the least cross reactive for all species tested The mAbs anti‐stress 70 N27, BRM‐22, and 3a3 were more broadly cross reactive, but their binding specificities to stress 70 isoforms in the diverse species tested did not correlate with one another or follow taxonomic lines The polyclonal anti‐stress 70 antibody reacted to proteins in the 70 to 74 kDa range in all fish examined and in most invertebrates When a polyclonal antibody (pAb) raised against cpn60 from a moth was used as a probe, specific binding was observed with proteins in the 60 to 64 kDa range in all fish examined and in most invertebrates However, the size and number of isoforms that reacted with the pAb were species specific These data suggest that these two major stress protein families are less highly conserved in invertebrates and fish than in mammals Therefore, to minimize misinterpretation when using antibodies in heterologous assays with species in which the stress response has not been well characterized, it is important to determine which isoforms of stress 70 react with a particular antibody and to take into account the differential regulation of each member of this multigene family

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Interleukin-1β induces the expression of HSP70, heme oxygenase and Mn-SOD in FACS-purified rat islet β-cells, but not in α-cells

et al. 1995

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The characterization and use of different antibodies against the hsp70 major heat shock protein family for the development of an immunoassay

Cited by 11 publications

References 18 publications

Differences in the Expression of Heat-Shock Proteins and Antioxidant Enzymes between Human and Rodent Pancreatic Islets: Implications for the Pathogenesis of Insulin-Dependent Diabetes Mellitus

Differences in the Expression of Heat-Shock Proteins and Antioxidant Enzymes between Human and Rodent Pancreatic Islets: Implications for the Pathogenesis of Insulin-Dependent Diabetes Mellitus

Specific cross‐reactivity of antibodies raised against two major stress proteins, stress 70 and chaperonin 60, in diverse species

Interleukin-1β induces the expression of HSP70, heme oxygenase and Mn-SOD in FACS-purified rat islet β-cells, but not in α-cells

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