Identification and analysis of all components of a gel retardation assay by combination with immunoblotting.

Demczuk, Stephen; Harbers, Matthias; Vennström, Björn

doi:10.1073/pnas.90.7.2574

Cited by 89 publications

(55 citation statements)

References 35 publications

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“…The nitrocellulose ®lter was subsequently immunoblotted with antibodies speci®c for pRb or p130, while the DE81 paper was autoradiographed to locate the radiolabeled oligonucleotide. This technique (termed shift/Western') provides an alternative means of identifying proteins associated with speci®c complexes (Demczuk et al, 1993). Using this technique, complexes I, II, and IV reacted with the pRb antibody ( Figure 4a).…”

Section: Identi®cation Of Prb Family Proteins By Immunoblottingmentioning

confidence: 99%

“…One mg of the appropriate antibody was preincubated with the cell extracts for 1 h on ice, after which the probe was added and the mixture was further incubated for 20 ± 30 min at room temperature before running the gel. After electrophoresis, the gel was blotted simultaneously onto nitrocellulose and DE-81 paper (Whatman), a procedure referred to as`shift Western' blotting (Demczuk et al, 1993). The transfer was carried out for 1 h, in a BioRad electroblotting unit with a cooling block, using chilled 25 mM Tris, 192 mM glycine and 20% v/v methanol.…”

Section: Electromobility Shift Assays and Shift-western Blottingmentioning

confidence: 99%

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pRb and p107 regulate E2F activity during lens fiber cell differentiation

et al. 1998

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During growth arrest and di erentiation, activity of the E2F family of transcription factors is inhibited by interactions with pRb and the related proteins, p107 and p130. To determine which members of the E2F and pRb families may contribute to growth arrest as lens epithelial cells di erentiate into ®ber cells, we examined the expression of individual E2F species and characterized the E2F protein complexes formed in rat lens epithelia and ®bers. RT/PCR detected all ®ve known members of the E2F family in lens epithelial cells, but only E2F-1, E2F-3, and E2F-5 in ®ber cells. Proteins extracted from lens epithelia of newborn rats formed at least two speci®c complexes with an E2F consensus oligonucleotide. Proteins from lens ®ber cells formed three speci®c complexes, one of which comigrated with an epithelial cell complex. Incubation of epithelial and ®ber cell extracts with an antibody speci®c for p107 demonstrated that two ®ber cell complexes and one epithelial cell complex contained p107. Although the remaining ®ber cell complex did not react with antibodies to pRb or p130 in this assay, a strong reaction with pRb antibody was observed when the electromobility shifted complexes were subsequently immunoblotted (shift/Western assay). Immunocytochemistry con®rmed that pRb protein is present in the nuclei of both epithelial cells and ®ber cells. Immunoblotting of whole cell extracts with pRb antibody showed multiple, phosphorylated forms of pRb in the epithelial cells, but predominantly hypophosphorylated pRb in the ®ber cells. None of the complexes formed with E2F were recognized exclusively by the p130 antibody, although the previously identi®ed p107 complexes reacted weakly. The absence of p130/E2F complexes was correlated with the presence of multiple ubiquitinated forms of p130, especially in the ®ber cells. Thus, although p130/E2F complexes are implicated in the terminal di erentiation of many cell types, in di erentiating lens ®ber cells pRb and p107 seem to be the primary regulators of E2F activity.

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Section: Identi®cation Of Prb Family Proteins By Immunoblottingmentioning

confidence: 99%

Section: Electromobility Shift Assays and Shift-western Blottingmentioning

confidence: 99%

pRb and p107 regulate E2F activity during lens fiber cell differentiation

et al. 1998

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“…Shift Western Blotting-To determine the relative stoichiometry of ParB present in the two forms of the partition complex, experiments were adapted from Demczuk et al (25) as follows. Gel mobility shift assays were performed as described above.…”

Section: Methodsmentioning

confidence: 99%

“…The resulting mixture of homo-and heterodimers (ParB/His-ParB mixture, lanes 5 and 9) was used in DNA binding assays. As controls, 25 g each of ParB and of His-ParB were independently denatured and renatured (in separate dialysis bags) as above and used in DNA binding assays (ParB, lanes 3 and 7; His-ParB, lanes 4 and 8). To isolate "His ϩ dimers" (dimers in which at least one monomer contains a polyhistidine tag), 15 g of the denatured/renatured mixture of ParB and His-ParB were purified over a 20-l nickel-agarose affinity chromatography column as described in Ref.…”

Section: Characterization Of Two Forms Of the Partition Complex-mentioning

confidence: 99%

“…To discriminate among these possibilities, we first performed shift Western blotting analysis (25) to measure the relative stoichiometry of ParB on parS in the two complexes. ParB-IHF DNA complexes were assembled on parS-132 DNA fragments, separated by electrophoresis, and then transferred to PVDF membranes, which retained both the DNA and proteins (see "Experimental Procedures").…”

Section: Characterization Of Two Forms Of the Partition Complex-mentioning

confidence: 99%

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Stoichiometry of P1 Plasmid Partition Complexes

Bouet

Surtees

Funnell

2000

Journal of Biological Chemistry

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The P1 plasmid prophage is faithfully partitioned by a high affinity nucleoprotein complex assembled at the centromere-like parS site. This partition complex is composed of P1 ParB and Escherichia coli integration host factor (IHF), bound specifically to parS. We have investigated the assembly of ParB at parS and its stoichiometry of binding. Measured by gel mobility shift assays, ParB and IHF bind tightly to parS and form a specific complex, called I ؉ B1. We observed that as ParB concentration was increased, a second, larger complex (I ؉ B2) formed, followed by the formation of larger complexes, indicating that additional ParB molecules joined the initial complex. Shift Western blotting experiments indicated that the I ؉ B2 complex contained twice as much ParB as the I ؉ B1 complex. Using mixtures of ParB and a larger polyhistidine-tagged version of ParB (His-ParB) in DNA binding assays, we determined that the initial I ؉ B1 complex contains one dimer of ParB. Therefore, one dimer of ParB binds to its recognition sequences that span an IHF-directed bend in parS. Once this complex forms, a second dimer can join the complex, but this assembly requires much higher ParB concentrations.

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Effect of in vivo administered 2,3,7,8-tetrachlorodibenzo-p-dioxin on DNA-binding activities of nuclear transcription factors in liver of guinea pigs

Ashida

Matsumura

1998

J. Biochem. Mol. Toxicol.

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Identification and analysis of all components of a gel retardation assay by combination with immunoblotting.

Cited by 89 publications

References 35 publications

pRb and p107 regulate E2F activity during lens fiber cell differentiation

pRb and p107 regulate E2F activity during lens fiber cell differentiation

Stoichiometry of P1 Plasmid Partition Complexes

Effect of in vivo administered 2,3,7,8-tetrachlorodibenzo-p-dioxin on DNA-binding activities of nuclear transcription factors in liver of guinea pigs

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