prsv luc  (Addgene inc)

Journal:

Article Title: Synergism between Calcium and Cyclic GMP in Cyclic AMP Response Element-Dependent Transcriptional Regulation Requires Cooperation between CREB and C/EBP-?

doi: 10.1128/MCB.23.12.4066-4082.2003

Figure Lengend Snippet: Synergistic activation of CRE- but not SRE-driven reporter genes by calcium and cGMP. UMR106 (A, B, and D) and C6 cells (C) were transiently transfected with the reporter plasmid pCRE-Luc (A to C) or pSRE-Luc (D) and with the control plasmid pRSV-βGal; cells were cotransfected with either empty vector (A, C, and D), G-kinase II expression vector (B to D), or a membrane-targeted G-kinase I construct (G-kinase I/II chimera; C) as described in Materials and Methods. Cells were maintained in low-serum medium for 24 h before they were treated for 8 h with 0.3 μM calcium ionophore A23187 (Ca++), 250 μM CPT-cGMP (cGMP), or both, as indicated. Luciferase and β-galactosidase activities were measured as described in Materials and Methods, and the ratio of luciferase to β-galactosidase activity of untreated cells was assigned a value of 1. Cotransfection of G-kinase II or the G-kinase I/II chimera had no significant effect in untreated cells.

Article Snippet: The control vectors pRSV-Luc and pRSV-βGal were described before ( 22 ), and the reporter pCRE-Luc (containing four tandem canonical CREs), pSRE-Luc, containing four copies of the fos SRE including the C/EBP-β binding site ( 46 ), and pAP1-Luc (containing four canonical AP-1 binding sites) were from Stratagene.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Construct, Luciferase, Activity Assay, Cotransfection

Journal:

Article Title: Synergism between Calcium and Cyclic GMP in Cyclic AMP Response Element-Dependent Transcriptional Regulation Requires Cooperation between CREB and C/EBP-?

doi: 10.1128/MCB.23.12.4066-4082.2003

Figure Lengend Snippet: Synergistic activation of CRE-dependent transcription by calcium and cGMP is prevented by dominant negative CREB and C/EBP constructs but not by a dominant negative Fos. (A and B) UMR106 cells were transfected with pCRE-Luc, pRSV-βGal, or G-kinase II vector as described in the legend to Fig. ​Fig.3;3; some cells were cotransfected with 40 ng of expression vector encoding the dominant negative A-CREB, A-C/EBP, A-Fos, or empty vector. Cells were treated for 8 h with 0.3 μM A23187 (Ca++), 250 μM CPT-cGMP (cGMP), or both (A), or cells were treated with 100 μM CPT-cAMP (B), as indicated. The ratio of luciferase to β-galactosidase activity was normalized as described in the legend to Fig. ​Fig.3.3. (C) Cells were transfected with pSRE-Luc, pRSV-βGal, and G-kinase II and cotransfected with empty vector (solid bars) or C/EBP-β (20 ng; open bars); some cells also received vector encoding A-C/EBP (40 ng) as indicated. (D) Cells were transfected with pAP1-Luc, pRSV-βGal, and G-kinase II and cotransfected with empty vector (solid bars) or JunB (20 ng; open bars); some cells also received vector encoding A-Fos (40 ng) as indicated.

Article Snippet: The control vectors pRSV-Luc and pRSV-βGal were described before ( 22 ), and the reporter pCRE-Luc (containing four tandem canonical CREs), pSRE-Luc, containing four copies of the fos SRE including the C/EBP-β binding site ( 46 ), and pAP1-Luc (containing four canonical AP-1 binding sites) were from Stratagene.

Techniques: Activation Assay, Dominant Negative Mutation, Construct, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay

Journal:

Article Title: Synergism between Calcium and Cyclic GMP in Cyclic AMP Response Element-Dependent Transcriptional Regulation Requires Cooperation between CREB and C/EBP-?

doi: 10.1128/MCB.23.12.4066-4082.2003

Figure Lengend Snippet: C/EBP-β enhances the effects of calcium and cGMP on a CRE-dependent reporter. (A) UMR106 cells were transfected with pCRE-Luc, pRSV-βGal, and G-kinase II as described in the legend to Fig. ​Fig.3;3; cells additionally received either empty vector (0 ng) or expression vector encoding C/EBP-β (6 ng and 12 ng). (B) Cells were transfected as in panel A except that pSRE-Luc was substituted for pCRE-luc and only 3 or 6 ng of C/EBP-β was cotransfected. Luciferase/β-galactosidase activity ratios were normalized as described in the legend to Fig. ​Fig.33.

Article Snippet: The control vectors pRSV-Luc and pRSV-βGal were described before ( 22 ), and the reporter pCRE-Luc (containing four tandem canonical CREs), pSRE-Luc, containing four copies of the fos SRE including the C/EBP-β binding site ( 46 ), and pAP1-Luc (containing four canonical AP-1 binding sites) were from Stratagene.

Techniques: Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay

Journal:

Article Title: Synergism between Calcium and Cyclic GMP in Cyclic AMP Response Element-Dependent Transcriptional Regulation Requires Cooperation between CREB and C/EBP-?

doi: 10.1128/MCB.23.12.4066-4082.2003

Figure Lengend Snippet: Decreased C/EBP-β phosphorylation in cGMP-treated cells. (A) C6 cells were transfected with an expression vector encoding C/EBP-β and either empty vector (left panel, lanes 1 to 5), G-kinase II (right panel, lanes 1 to 5) or a G-kinase I/II chimera (right panel, lanes 6 and 7); cells were incubated with 32PO4 for 4 h and treated for 1 h with buffer (lanes 1, 5, and 6), 0.3 μM A23187 (lane 2), 250 μM CPT-cGMP (lane 3), or both agents (lanes 4 and 7) as described in Materials and Methods. Cell lysates were subjected to immunoprecipitation with a rabbit anti-C/EBP-β antibody (lanes 1 to 4, 6, and 7) or control rabbit IgG (lane 5). Immunoprecipitates were analyzed by SDS-PAGE, electroblotting, and autoradiography (upper panel) and by blotting with a murine anti-C/EBP-β antibody (lower panel). The immunoglobulin heavy chain band is labeled Ig. (B) 32PO4 incorporation into C/EBP-β was quantitated by scanning densitometry of autoradiographs of three independent experiments performed as described for panel A; cells were transfected with C/EBP-β plus either empty vector (open bars) or G-kinase II (solid bars). (C) Cells were transfected with C/EBP-β and G-kinase II and labeled for 4 h in 32PO4-containing medium as described for panel A. Half of the cultures were treated with 0.3 μM A23187 and 250 μM CPT-cGMP for the indicated times, and 32PO4 incorporation into C/EBP-β was quantitated as described for panel B. Data are expressed as percentages of the level in untreated controls. (D) Cells transfected with C/EBP-β were labeled with 32PO4 for 4 h and either left untreated (lanes 1 and 2) or treated for 1 h with 5 mM sodium valproate (lane 3); cell lysates were processed as described for panel A (lane 1, control IgG; lanes 2 and 3, anti-C/EBP-β antibody). (E) Cells were transfected with pCRE-Luc, pRSV-βGal, and either empty vector (E.V.) or 12 ng of C/EBP-β vector; cultures were either left untreated (open bars) or treated with 5 mM sodium valproate for 8 h (solid bars). Luciferase activity was normalized to β-galactosidase activity as described for Fig. ​Fig.33.

Article Snippet: The control vectors pRSV-Luc and pRSV-βGal were described before ( 22 ), and the reporter pCRE-Luc (containing four tandem canonical CREs), pSRE-Luc, containing four copies of the fos SRE including the C/EBP-β binding site ( 46 ), and pAP1-Luc (containing four canonical AP-1 binding sites) were from Stratagene.

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Immunoprecipitation, SDS Page, Autoradiography, Labeling, Luciferase, Activity Assay

Journal:

Article Title: Synergism between Calcium and Cyclic GMP in Cyclic AMP Response Element-Dependent Transcriptional Regulation Requires Cooperation between CREB and C/EBP-?

doi: 10.1128/MCB.23.12.4066-4082.2003

Figure Lengend Snippet: Effect of calcium and cGMP on CREB phosphorylation and effect of Cam-kinase and MAP kinase inhibitors on calcium- and cGMP-stimulated transcription. (A) UMR106 cells (left panel) and C6/GKII.1 cells (right panel) were treated for 1 h with 0.3 μM A23187 (lane 2), 250 μM CPT-cGMP (lane 3), or both (lane 4). Equal amounts of cell extracts were analyzed by SDS-PAGE and Western blotting with an antibody specific for Ser133-phosphorylated CREB (pCREB, upper panel) and an antibody that recognizes CREB irrespective of its phosphorylation status (CREB, lower panel). The phospho-CREB antibody cross-reacts with phosphorylated ATF-1 and probably CREM (64). (B) UMR106 cells were transfected with pCRE-Luc, pRSV-βGal, and G-kinase II and treated with A23187 (Ca++), CPT-cGMP (cGMP), or both, as described in the legend to Fig. ​Fig.3.3. At 24 h before harvesting, some cells were treated with 0.1% dimethyl sulfoxide (vehicle; DMSO), 10 μM U0126, 10 μM SB20358, or 10 μM KN62. Reporter gene activities were normalized as described for Fig. ​Fig.33.

Article Snippet: The control vectors pRSV-Luc and pRSV-βGal were described before ( 22 ), and the reporter pCRE-Luc (containing four tandem canonical CREs), pSRE-Luc, containing four copies of the fos SRE including the C/EBP-β binding site ( 46 ), and pAP1-Luc (containing four canonical AP-1 binding sites) were from Stratagene.

Techniques: SDS Page, Western Blot, Transfection

Journal:

Article Title: Synergism between Calcium and Cyclic GMP in Cyclic AMP Response Element-Dependent Transcriptional Regulation Requires Cooperation between CREB and C/EBP-?

doi: 10.1128/MCB.23.12.4066-4082.2003

Figure Lengend Snippet: Calcium and cGMP synergistically increase the transactivation potential of full-length Gal4-CREB but not of Gal4-CREB-Δbzip; effect of A-C/EBP. (A) UMR106 cells were transfected with the reporter plasmid pGAL4-Luc, pRSV-βGal, and G-kinase II; cells were cotransfected with a vector encoding either full-length Gal4-CREB or Gal4-CREB-Δbzip. Cells were treated with A23187 (Ca++) and/or CPT-cGMP (cGMP), and reporter gene activities were measured as described in the legend to Fig. ​Fig.3.3. The luciferase/β-galactosidase activity ratio of untreated cells transfected with full-length Gal4-CREB was assigned a value of 1. We transfected different amounts of vector encoding full-length Gal4-CREB (15 ng) or Gal4-CREB-Δbzip (5 ng) to produce similar reporter gene activities in untreated cells. When the same amount of each vector was transfected, the activity of Gal4-CREB-Δbzip was 2.8- ± 0.4-fold higher than the activity of full-length Gal4-CREB (not shown). (B) In parallel experiments, cells were transfected with 0.1 μg, 0.3 μg, or 1 μg of full-length Gal4-CREB or Gal4-CREB-Δbzip, as indicated, to examine the expression levels of both constructs by Western blotting with an antibody specific for the Gal4 DNA-binding domain. (C) UMR106 cells were transfected with full-length Gal4-CREB, pGAL4-Luc, pRSV-βGal, and G-kinase II as described for panel A; cells were cotransfected with either empty vector or 40 ng of expression vector encoding A-CREB, A-C/EBP, or A-Fos. Cells were treated, and luciferase/β-galactosidase activity ratios were determined as described for panel A.

Article Snippet: The control vectors pRSV-Luc and pRSV-βGal were described before ( 22 ), and the reporter pCRE-Luc (containing four tandem canonical CREs), pSRE-Luc, containing four copies of the fos SRE including the C/EBP-β binding site ( 46 ), and pAP1-Luc (containing four canonical AP-1 binding sites) were from Stratagene.

Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Construct, Western Blot, Binding Assay

Journal:

Article Title: Synergism between Calcium and Cyclic GMP in Cyclic AMP Response Element-Dependent Transcriptional Regulation Requires Cooperation between CREB and C/EBP-?

doi: 10.1128/MCB.23.12.4066-4082.2003

Figure Lengend Snippet: C/EBP-β enhances the transactivation potential of Gal4-CREB and Gal4-CREB-Δbzip. (A) UMR106 cells were transfected with pGAL4-Luc, pRSV-βGal, and Gal4-ATF-1 (100 ng), Gal4-CREB (15 ng), or Gal4-CREB-Δbzip (5 ng) as indicated; cells were cotransfected with either empty vector, 100 ng of expression vector encoding p20, 100 ng of p20 fused to the activation domain of VP16, or 12 ng of full-length C/EBP-β (p35). Different amounts of vector encoding p20, p20-VP16, and p35 were used because Western blotting with an antibody specific for the C terminus of C/EBP-β showed that the p20 and p20-VP16 vectors expressed about eightfold-lower protein levels than the same amount of p35 C/EBP-β vector (not shown). The luciferase/β-galactosidase activity ratio of untreated cells transfected with full-length Gal4-CREB was assigned a value of 1. (B and C) C6 cells were transfected with pGAL4-Luc, pRSV-βGal, G-kinase II, and either full-length Gal4-CREB (15 ng, panel B) or pGal4-CREBΔbzip (5 ng, panel C); cells were cotransfected with either empty vector (control) or 12 ng of expression vector encoding C/EBP-β. Cells were treated with A23187 (Ca++) and/or CPT-cGMP (cGMP), and luciferase/β-galactosidase activity ratios were determined as described in the legend to Fig. ​Fig.33.

Article Snippet: The control vectors pRSV-Luc and pRSV-βGal were described before ( 22 ), and the reporter pCRE-Luc (containing four tandem canonical CREs), pSRE-Luc, containing four copies of the fos SRE including the C/EBP-β binding site ( 46 ), and pAP1-Luc (containing four canonical AP-1 binding sites) were from Stratagene.

Techniques: Transfection, Plasmid Preparation, Expressing, Activation Assay, Western Blot, Luciferase, Activity Assay