Rapid detection of octamer binding proteins with ‘mini extracts’, prepared from a small number of cells

Schreiber, Edgar; Matthias, Patrick; Müller, Michael M.; Schaffner, Walter

doi:10.1093/nar/17.15.6419

Cited by 4,030 publications

(2,593 citation statements)

References 2 publications

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“…Nuclear extracts were pre ared according to the method of Schreiber et al [44]. Cells (4 x 10 ) were resuspended in 400 p1 of ice-cold buffer A (10 mM HEPES (pH 7.9), 10 mM KCI, 0.1 mM EDTA, 0.1 EGTA, 1 mM DTT, and 0.5 mM PMSF) and were then lysed with 25 p1 of 10% Nonidet P-40.…”

Section: Nuclear Extractsmentioning

confidence: 99%

Activation of NF-KB signalling and TNFα-expression in THP-1 macrophages by TiAlV- and polyethylene-wear particles

et al. 2005

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Wear particles are believed to induce periprosthetic inflammation which contributes to periprosthetic osteolysis. TNFu plays a pivotal role in the pathogenesis of this process. The molecular mechanisms leading to the development of periprosthetic inflammation with upregulated TNFa expression in monocytic cells in response to different wear particles have yet to be defined. 1n:this study we evaluated the effects of polyethylene-and TiAIV-particles on activation of NF-KB signalling pathways and TNFa biosynthesis and release in monocytic cells with respect to periprosthetic osteoclastogenesis. THP-1 monocytic cells were differentiated to macrophage-like cells and exposed to LPS-detoxified polyethylene and prosthesis-derived TiAlV-particles. TNFa release was analyzed in culture supernatant by ELISA. NF-KB activation was examined by electrophoretic mobility shift assay (EMSA), and NF-KB target promoter activities including transactivation of the TNFa promoter were determined by luciferase reporter gene assays. Differentiated THP-1 macrophages were exposed to increasing numbers of particles for 0, 60, 180 and 360 min. Both, polyethylene-and TiAIV-particles induced a significant activation of both NF-KB and TNFa promoters at 180 min. A significant TNFu release was detected after 360 min exposure to polyethylene-and TiAlV-particles in a dose dependent manner. In comparison, LPS induced a much greater activation of NF-KB and TNFa promoters, and TNFa secretion into the supernatant was strongly induced. These results provide evidence that induction of the NF-KB signal transduction pathway in macrophages plays a major role in initiating and mediating the inflammatory response leading to periprosthetic osteolysis.

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Section: Nuclear Extractsmentioning

confidence: 99%

Activation of NF-KB signalling and TNFα-expression in THP-1 macrophages by TiAlV- and polyethylene-wear particles

et al. 2005

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“…Forty-eight hours post transfection, cells were harvested by trypsinisation, and nuclear extracts were prepared as described elsewhere (Schreiber et al, 1989).…”

Section: Preparation Of Nuclear Extractsmentioning

confidence: 99%

Transcriptional modulation of the anti-apoptotic protein BCL-XL by the paired box transcription factors PAX3 and PAX3/FKHR

et al. 2000

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The aberrant expression of the transcription factors PAX3 and PAX3/FKHR associated with rhabdomyosarcoma (RMS), solid tumors displaying muscle cell features, suggests that these proteins play an important role in the pathogenesis of RMS. We could previously demonstrate that one of the oncogenic functions of PAX3 and PAX3/FKHR in RMS is protection from apoptosis. BCL-XL is a prominent anti-apoptotic protein present in normal skeletal muscle and RMS cells. In the present study, we establish that BCL-XL is transcriptionally modulated by PAX3 and PAX3/FKHR, since enhanced expression of both PAX proteins stimulates transcription of endogenous BCL-XL mRNA in a cell type speci®c manner. Further, we present evidence that both PAX3 and PAX3/FKHR can transcriptionally activate the Bcl-x gene promoter in cotransfection assays. Using electrophoretic mobility shift assays, an ATTA binding site for PAX3 and PAX3/FKHR could be localized in the upstream promoter region (position 742 to 739). Finally, ectopic overexpression of either PAX3, PAX3/FKHR or BCL-XL can rescue tumor cells from apoptosis induced by antisense treatment. These results suggest that at least part of the anti-apoptotic e ect of PAX3 and PAX3/FKHR is mediated through direct transcriptional modulation of the prominent antiapoptotic protein BCL-XL.

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“…Electrophoretic Mobility Shift Assay (EMSA) was performed as described previously (Rutberg et al, 1996) using 3 mg of nuclear extract (Schreiber et al, 1989) per reaction. DNA binding complexes were visualized using a 32 P-labeled probe bearing a single copy of the consensus AP-1 (5'-cgcttgaTGAGTCAgccggaa-3') or CRE (5'-agagattgccT-GACGTCA-gagagctag-3') target sequences.…”

Section: Electrophoretic Mobility Shift Assaymentioning

confidence: 99%

CRE DNA binding proteins bind to the AP-1 target sequence and suppress AP-1 transcriptional activity in mouse keratinocytes

et al. 1999

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Previously, we have shown that nuclear extracts from cultured mouse keratinocytes induced to di erentiate by increasing the levels of extra-cellular calcium contain Fra-1, Fra-2, Jun B, Jun D and c-Jun proteins that bind to the AP-1 DNA binding sequence. Despite this DNA binding activity, AP-1 reporter activity was suppressed in these cells. Here, we have detected the CREB family proteins CREB and CREMa as additional participants in the AP-1 DNA binding complex in di erentiating keratinocytes. AP-1 and CRE DNA binding activity correlated with the induction of CREB, CREMa and ATF-1 and CREB phosphorylation at ser 133 (ser 133 phospho-CREB) in the transition from basal to di erentiating keratinocytes, but the activity of a CRE reporter remained unchanged. In contrast, the CRE reporter was activated in the presence of the dominantnegative (DN) CREB mutants, KCREB and A-CREB, proteins that dimerize with CREB family members and block their ability to bind to DNA. The increase in CRE reporter activity in the presence of these mutants suggests that CRE-mediated transcriptional activity is suppressed in keratinocytes through protein-protein interactions involving a factor that dimerizes with the CREB leucine zipper. In experiments where the A-CREB mutant was co-transfected with an AP-1 reporter construct, transcriptional activity was also increased indicating that a CREB family member binds AP-1 sites and represses AP-1 transcriptional activity as well. Exogenous expression of the transcriptional repressor CREMa down-regulated both CRE and AP-1 reporters in keratinocytes suggesting that this factor may contribute to the suppression of AP-1 transcriptional activity observed in di erentiating keratinocytes.

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Rapid detection of octamer binding proteins with ‘mini extracts’, prepared from a small number of cells

Cited by 4,030 publications

References 2 publications

Activation of NF-KB signalling and TNFα-expression in THP-1 macrophages by TiAlV- and polyethylene-wear particles

Activation of NF-KB signalling and TNFα-expression in THP-1 macrophages by TiAlV- and polyethylene-wear particles

Transcriptional modulation of the anti-apoptotic protein BCL-XL by the paired box transcription factors PAX3 and PAX3/FKHR

CRE DNA binding proteins bind to the AP-1 target sequence and suppress AP-1 transcriptional activity in mouse keratinocytes

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