Chapter 27 Two-Dimensional Polyacrylamide Gel Electrophoretic Fractionation

O’Farrell, Patrick H.; O'Farrell, Patricia Z.

doi:10.1016/s0091-679x(08)60116-8

Cited by 130 publications

(53 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The residual pellet was washed twice with water, and the supernatants were pooled with the solubilized sample, lyophilized, and resuspended in 50 µL of water. Two-dimensional gel electrophoresis was performed according to the method of O'Farrell by Kendrick Labs Inc. (Madison, Wisconsin, USA) (12). Five microliters of the solubilized immunoprecipitate was subjected to isoelectric focusing in glass tubes of 2-mm inner diameter using 2% (pH 4-8) ampholines (BDH; Hoefer Scientific Instruments, San Francisco, California, USA) for 9,600 volt-hours.…”

Section: Figurementioning

confidence: 99%

Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin

Topham¹,

Kawachi²,

Haydar³

et al. 1999

J. Clin. Invest.

158

100

View full text Add to dashboard Cite

Section: Figurementioning

confidence: 99%

Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin

Topham¹,

Kawachi²,

Haydar³

et al. 1999

J. Clin. Invest.

158

100

View full text Add to dashboard Cite

“…Modulation of the synthesis of proteins involved in cell adhesion or protection from nonspecific injury are among several possible mechanisms by which 1,25-(OH)2D3 could maintain monocyte adherence. We therefore analyzed the cellular proteins synthesized by these cells by two-dimensional gel electrophoresis (16,17) and found an increase in the synthesis of several proteins in 1,25-(OH)2D3-treated monocytes. Moreover, inhibitors of protein synthesis abolished the effects of 1,254OH)2D3 on maintenance of monocyte adherence.…”

Section: Introductionmentioning

confidence: 99%

1,25-Dihydroxyvitamin D3 maintains adherence of human monocytes and protects them from thermal injury.

Polla¹,

Healy²,

Amento³

et al. 1986

J. Clin. Invest.

View full text Add to dashboard Cite

Adherence to a substratum is a characteristic feature of monocyte-macrophages which may be required for several effector functions. Human peripheral blood monocytes selected by adherence were found to readhere preferentially at 1 h to fibronectin or to a biological matrix. There was then a progressive decrease in the number of adherent cells, and by 48 h only 8-20% of monocytes remained adherent. This loss of adherence occurred while monocytes remained viable by criteria such as exclusion of trypan blue or release of lactate dehydrogenase.1,25-dihydroxyvitamin D3 (1,254OH)2D3) maintained the adherence of cultured monocytes to tissue culture plastic as well as to the biological matrix. This effect was concentration-and time-dependent, and suppressed by inhibitors of protein synthesis. Cellular proteins were labeled after incubation with [35Sjmethionine. Analysis by two-dimensional gel electrophoresis revealed increased labeling of several distinct proteins in 1,25-(OH)2D3-treated monocytes compared with control monocytes. The increased loss of adherence and decreased overall protein synthesis observed in monocytes incubated at 45°C was partially prevented by preincubation of the cells with 1,25-OH)2D3. We further evaluated the effects of thermal stress and 1,254OH)2D3 on protein synthesis by monocytes, and found that 1,254OH)D3 increased the synthesis of heat shock proteins, protected normal protein synthesis, and increased the rate of recovery of normal protein synthesis after the thermal stress.These observations suggest that 1,25-(OH)2D3 influences monocytes by preserving the synthesis of proteins, including those critical for the maintenance of cell adherence.

show abstract

“…ing to the method of O'Farrell (O'Farrell & O'Farrell, 1978), using a Hoeffer Scientific Instruments gel apparatus. Prior to electrophoresis, protein samples were quantitated by a BCA assay (Pierce) and specific activities were determined following TCA precipitation and liquid scintillation counting.…”

mentioning

confidence: 99%

The identification of heme oxygenase as a major hypoxic stress protein in Chinese hamster ovary cells

Murphy

Laderoute²,

Short³

et al. 1991

Br J Cancer

123

View full text Add to dashboard Cite

Summary Chronic hypoxia increases the expression of a set of stress proteins (oxygen regulated proteins or ORPs) which is implicated in the development of drug resistance and radiation sensitivity in tumour cells. Five major ORPs have been documented, and two, ORP 80 and ORP 100, are considered to be identical to the glucose regulated stress proteins GRP78 and GRP94, respectively. We report here that ORP 33 is a form of the heme catabolic enzyme, heme oxygenase, using evidence obtained from northern blotting, two-dimensional polyacrylamide gel electrophoresis and western analysis. Heme oxygenase is believed to be an important component of the cellular response to oxidative stress. The significance of heme oxygenase as a hypoxiainduced stress protein is discussed.Hypoxia is an important environmental stress encountered in some solid tumours that can influence the effectiveness of radiation, hyperthermia and chemo-therapy (Coleman, 1988;Heacock & Sutherland, 1990). Poor vascularisation of tumours is common, and this results in impaired delivery of oxygen, glucose and other nutrients to cells distant from blood vessels, as well as inefficient removal of metabolic wastes (Sutherland, 1988). Among the many changes in tumour cellular physiology that occur under hypoxic stress, it has been observed that a group of proteins, called oxygen regulated proteins or ORPs, can be induced to undergo enhanced rates of synthesis depending on the severity and duration of the stress (Heacock & Sutherland, 1990;Anderson et al., 1979;Heacock & Sutherland, 1986). Moreover, the kinetics of the development of resistance to the drug adriamycin in vitro have been reported to correlate with the enhanced expression of some of these ORPs Subjeck & Shyy, 1986). Another phenotype associated with chronic hypoxia is enhanced radiation sensitivity following reoxygenation. Although low oxygen is known to give rise to radiation resistance, through a diminishment of the phenomenon known as the oxygen effect (von Sonntag, 1987), upon reoxygenation chronically hypoxic cells exhibit enhanced sensitivity to gamma radiation (Kwok & Sutherland, 1989a;Kwok & Sutherland, 1989b). The mechanistic basis of this effect is not understood, although it may be a consequence of the stress caused by reoxygenation rather than by hypoxia per se.The molecular weights assigned to five major ORPs are 260, 150, 100, 80 and 33 kilodaltons (Heacock & Sutherland, 1986 In experiments designed to determine a time course for the induction of heme oxygenase under hypoxia, the chambers were opened, the plates were placed on ice, the media was removed by aspiration and the cells were immediately lysed as above by the addition of cold lysis buffer.Polyacrylamide gel electrophoresis and western blotting The labelled Triton soluble proteins were analyzed by twodimensional SDS-polyacrylamide gel electrophoresis accordCorrespondence: R.M. Sutherland.

show abstract

Chapter 27 Two-Dimensional Polyacrylamide Gel Electrophoretic Fractionation

Cited by 130 publications

References 10 publications

Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin

Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin

1,25-Dihydroxyvitamin D3 maintains adherence of human monocytes and protects them from thermal injury.

The identification of heme oxygenase as a major hypoxic stress protein in Chinese hamster ovary cells

Contact Info

Product

Resources

About