pgas ta luc firefly luciferase reporter  (TaKaRa)

Journal: Cellular signalling

Article Title: E3 ubiquitin ligase NKLAM ubiquitinates STAT1 and positively regulates STAT1-mediated transcriptional activity

doi: 10.1016/j.cellsig.2016.08.014

Figure Lengend Snippet: NKLAM positively affects STAT1-mediated transcriptional activity. A) WT (black column) and NKLAM-KO (white column) BMDM were nucleofected with pGAS-luc plasmid then stimulated with 100 U/mL IFNγ for 6 h and assayed for luciferase activity; *p < 0.05; n = 5. B–C) WT (black column) and NKLAM-KO (white column) BMDM were treated with 100 U/mL IFNγ for 3 or 6 h. The expression of iNOS and CCL5 was determined by quantitative RT-PCR. The fold change in mRNA levels (mean ± SEM) is expressed relative to untreated cultures (solid line set at 1). Graph represents at least 3 independent experiments. *p < 0.05; n = 3.

Article Snippet: WT or NKLAM-KO macrophages (4 × 10 6 ) were mixed with 3 μg of the luciferase reporter plasmid pGAS-luc and suspended in nucleofection solution T (Lonza).

Techniques: Activity Assay, Plasmid Preparation, Luciferase, Expressing, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Restoration of IFNγR Subunit Assembly, IFNγ Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol

doi: 10.1371/journal.ppat.1002229

Figure Lengend Snippet: A, Measurements of FA value of RAW 264.7 cells left untreated or treated with 10 µg/ml of purified LPG for varying time periods. B , RAW 264.7 cells left untreated or treated with 10 µg/ml of purified LPG for 18 hrs (time equivalent of total parasite-MØ interaction of 6 hrs of initial attachment and 12 hours of infection establishment) were subjected to membrane harvestation. Gradient fractions were purified as described in . Equal volume of fractions from each optiprep gradient were analyzed by dot blot for the presence of GM1, IFNγR1, LPG by using the appropriate antibodies and CTxB-HRP. C, IFNγ induced bioactivity in LPG treated MØs was determined by reporter assay. RAW 264.7 cells were transfected with pCIS-CK negative control vector or IFNγ driven pGAS reporter plasmid. 12 hrs after transfection, cells were either left untreated or treated with mBCD (5 mM, 30 min, 37°C) and /or treated with 10 µg/ml of LPG for 18 hours. Treated cells were then replenished or not with CH-Lipo. Subsequently treated cells were stimulated with or without 100 U/ml of rIFNγ eight hours before final harvesting for luciferase activity determination in cell lysates. The data represents the relative luciferase activity normalized to per microgram of protein content. Data are representative of three independent experiments.

Article Snippet: RAW 264.7 cells were transfected with the pGAS cis-reporter plasmids or the pCIS-CK negative control plasmid by electroporation by Genepulser X cell (Bio-Rad, Hercules, CA).

Techniques: Purification, Infection, Dot Blot, Reporter Assay, Transfection, Negative Control, Plasmid Preparation, Luciferase, Activity Assay

Journal: PLoS Pathogens

Article Title: Restoration of IFNγR Subunit Assembly, IFNγ Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol

doi: 10.1371/journal.ppat.1002229

Figure Lengend Snippet: Infected RAW 264.7 cells were left untreated or treated with CH-Lipo or AN-Lipo or DPPC-Lipo followed by stimulation with 100 U/ml of rIFNγ for 24 hours at 4 th hour post infection ( A, B, C ) or 12 th hour post infection ( A', B', C' ) and the number of intracellular amastigotes per MØ, % infected MØs and nitrite concentration (µM) were determined. Data are representative of three independent experiments. D , Pretreatment with inducible nitric oxide synthase inhibitor blocks IFNγ induced leishmanicidal effect in CH-Lipo treated infected MØs. RAW 264.7 cells were infected with LD at m.o.i of 10, for 6 hours. MØs were then washed and either treated with CH-Lipo or left untreated as infected control. Subsequently, at indicated timepoints postinfection, MØs were stimulated with or without 100 U/ml of rIFNγ for 24 hours in presence or absence of 5 µM L-NMMA. Cell free supernatant was harvested for NO measurement. Data of Nitrite concentration (µM) is representative of three independent experiments. The amastigote numbers of the parallel sets treated similarly was demonstrated in D' . E , Enhanced IFNγ-bioactivity in CH-Lipo replenished MØs as determined by reporter assay. RAW 264.7 cells were transfected with pCIS-CK negative control vector or IFNγ driven pGAS reporter plasmid. 12 hrs after transfection, cells were either left untreated or treated with mBCD (5 mM, 30 min, 37°C) or infected with LD. Treated cells were then replenished or not with CH-Lipo. Subsequently, mBCD treated cells with or without CH-Lipo reloading were stimulated with or without 100U/ml of rIFNγ eight hours before final harvesting for luciferase activity determination in cell lysates. While the infected cells with or without CH-Lipo treatment were stimulated or not with 100 U/ml of rIFNγ at indicated timepoints post infection. Eight hours later, luciferase activities in cell lysates were measured as described in . The data represents the relative luciferase activity normalized to per microgram of protein content. Data is representative of three independent experiments.

Article Snippet: RAW 264.7 cells were transfected with the pGAS cis-reporter plasmids or the pCIS-CK negative control plasmid by electroporation by Genepulser X cell (Bio-Rad, Hercules, CA).

Techniques: Infection, Concentration Assay, Reporter Assay, Transfection, Negative Control, Plasmid Preparation, Luciferase, Activity Assay

Journal:

Article Title: Cyclin-dependent kinase 5 phosphorylates signal transducer and activator of transcription 3 and regulates its transcriptional activity

doi: 10.1073/pnas.0307606100

Figure Lengend Snippet: Cdk5 regulated the STAT3 DNA-binding and transcriptional activity in vitro and in vivo. (A) Blockade of Cdk5 activity by Ros inhibited the NRG-induced increase in DNA binding of STAT3 in C2C12 myotubes. (Lower) Total STAT3. (Right) DNA-binding activity of STAT3 α and β was quantitated and expressed as a ratio of that in control myotubes pretreated with DMSO. Each value represented the mean ± SEM. of three representative experiments; *, P < 0.005. (B) Northern blot analysis of c-fos and junB in primary muscle cultures prepared from E18.5 wild-type (+/+) or Cdk5 null (-/-) embryos with (+) or without (-) NRG treatment. (Upper) c-fos. (Lower) junB. (C) DNA binding of STAT3 in E18.5 Cdk5 wild-type (+/+) and mutant (-/-) muscle (Left) and quantitation (Right). DNA-binding activity of STAT3 α and β was quantitated and expressed as a ratio of that in wild-type muscle. Each value represented the mean ± SEM. of three representative experiments; *, P < 0.005. (D) Northern blot analysis of fibronectin and muscle creatine kinase (MCK) in wild-type (+/+) and Cdk5-deficient (-/-) muscle of E18.5 embryos. MCK served as an equal loading control. (E) Overexpression of p35 increased the transcriptional activity of STAT3 in primary chick myotubes in a dose-dependent manner. Primary chick myotubes were transiently transfected with a STAT3 (pSTAT3-TA-Luc) or STAT1 (pGAS-TA-Luc) reporter gene construct and an internal control plasmid (β-gal-pCMV) with or without p35 or dnCdk5. The ratio of DNAs used for transfection was as depicted on the x axis. Luciferase activity was measured and normalized against the β-gal activity in the samples. Promoter activity was expressed as the ratio of luciferase activity in cells transfected with different combinations of p35 and dnCdk5 relative to that transfected with the empty vector. The data represented the mean ± SEM, n = 5. (F) Overexpression of p35 increased the transcriptional activity of c-fos. c-fos promoter-luciferase assay was performed as described in E with pSTAT3-TA-Luc (pSTAT3) as a positive control. Preincubation with Ros reduced Cdk5/p35-mediated transcriptional activity of pSTAT3 or c-fos. Overexpression of p35 could not increase the luciferase reporter activity under the control of STAT1 enhancer-responsive element (pGAS). The data represented mean ± SEM, n = 3. (G) The increase of STAT3 transcriptional activity depends on Ser-727 phosphorylation of STAT3. Primary muscle cultures were transfected with indicated expression constructs and luciferase assay was performed as in E; mean ± SEM, n = 3.

Article Snippet: Transfection mixtures consisted of a mixture of pSTAT3-TA-Luc, pGL3-c-fos, or pGAS reporter together with p35 or the following cDNA constructs (dnCdk5, STAT3, or STAT3M) and β-galactosidase (βgal)pCMV constructs (Clontech).

Techniques: Binding Assay, Activity Assay, In Vitro, In Vivo, Northern Blot, Mutagenesis, Quantitation Assay, Over Expression, Transfection, Construct, Plasmid Preparation, Luciferase, Positive Control, Expressing