Journal: BMC Immunology

Article Title: Forced IFIT-2 expression represses LPS induced TNF-alpha expression at posttranscriptional levels

doi: 10.1186/1471-2172-9-75

Figure Lengend Snippet: Protein expression of IFIT-2 in RAW 264 .7 macrophages. RAW 264.7 cells were stimulated with (A) IFN-β (50 U/ml), LPS (1 μg/ml), Pam3Cys (1 μg/ml) or IFN-γ (50 ng/ml), pretreated with GIR-208 or MAR1 prior to (C) LPS, (D) Pam3Cys or IFN-γ stimulation for indicated time periods and IFIT-2 and β-actin expression was detected by immunoblots. (B) RAW 264.7 cells were stimulated with or without IFN-β, staining was performed using anti-IFIT-2 and a secondary Cy2-labeled goat anti-rabbit antibody (green), Phalloidin-Tritc (red) and DAPI (blue) and detected by immunofluorescence. Big pictures; IFIT-2 expression, small pictures overlay of phalloidin and DAPI staining. At least three independent experiments with similar results with exception of B (two experiments).

Article Snippet: For staining a polyclonal anti-IFIT-2 and goat-anti-rabbit IgG-PE antibody (BD Biosciences) were used.

Techniques: Expressing, Western Blot, Staining, Labeling, Immunofluorescence

Journal: BMC Immunology

Article Title: Forced IFIT-2 expression represses LPS induced TNF-alpha expression at posttranscriptional levels

doi: 10.1186/1471-2172-9-75

Figure Lengend Snippet: Protein expression of stably transfected RAW-IFIT-2 cells . IFIT-2 expression was detected in RAW 264.7 and RAW-IFIT-2 cells by (A) immunoblot or (B) intracellular staining and flow cytometry (at least two experiments).

Article Snippet: For staining a polyclonal anti-IFIT-2 and goat-anti-rabbit IgG-PE antibody (BD Biosciences) were used.

Techniques: Expressing, Stable Transfection, Transfection, Western Blot, Staining, Flow Cytometry

Journal: BMC Immunology

Article Title: Forced IFIT-2 expression represses LPS induced TNF-alpha expression at posttranscriptional levels

doi: 10.1186/1471-2172-9-75

Figure Lengend Snippet: Forced IFIT-2 expression modulates protein expression . RAW264.7 and RAW-IFIT-2 cells were stimulated with 1 μg/ml or as stated LPS for 24 h or as stated and (A) TNF-α-, (B) IL-6, (C) MIP-2 or (D) VEGF secretion was determined by ELISA. Significant differences (two-way ANOVA or t-test) were found for TNF-α, IL-6 and MIP-2 (p < 0.001). Three independent experiments with similar results.

Article Snippet: For staining a polyclonal anti-IFIT-2 and goat-anti-rabbit IgG-PE antibody (BD Biosciences) were used.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: BMC Immunology

Article Title: Forced IFIT-2 expression represses LPS induced TNF-alpha expression at posttranscriptional levels

doi: 10.1186/1471-2172-9-75

Figure Lengend Snippet: EGR-1 and IFIT-1 expression in RAW-IFIT-2 cells after LPS stimulation. Immunoblots were performed after LPS stimulation of cells for detection of (A) EGR-1 and Histone H3 in the nucleus or (B) IFIT-1 and actin in whole cell lysates. Three independent experiments with similar results.

Article Snippet: For staining a polyclonal anti-IFIT-2 and goat-anti-rabbit IgG-PE antibody (BD Biosciences) were used.

Techniques: Expressing, Western Blot

Journal: BMC Immunology

Article Title: Forced IFIT-2 expression represses LPS induced TNF-alpha expression at posttranscriptional levels

doi: 10.1186/1471-2172-9-75

Figure Lengend Snippet: Impact of IFIT-2 on LPS mediated signal transduction . RAW and RAW-IFIT-2 cells were stimulated with LPS (1 μg/ml) for indicated time periods. Immunoblots were performed for total and phosphorylated proteins as indicated. Three independent experiments with similar results.

Article Snippet: For staining a polyclonal anti-IFIT-2 and goat-anti-rabbit IgG-PE antibody (BD Biosciences) were used.

Techniques: Transduction, Western Blot

Journal: BMC Immunology

Article Title: Forced IFIT-2 expression represses LPS induced TNF-alpha expression at posttranscriptional levels

doi: 10.1186/1471-2172-9-75

Figure Lengend Snippet: TNF-α and IFIT-1 mRNA expression in RAW-IFIT-2 cells . RAW and RAW-IFIT-2 cells were stimulated with LPS (1 μg/ml) for indicated time periods and mRNA expression was detected for (A) TNF-α and (B) IFIT-1 by Real Time RT-PCR normalized to the house keeping gene RPL8. Data are shown as fold increased expression compared to unstimulated cells. Asterisks indicate significant differences p < 0.005 (Two-Way Anova). (C) Cells were stimulated for two hours with LPS and then treated for indicated time periods with actinomycin D. TNF-α and RPL8 mRNA expression was determined by Real-time RT PCR. Data are shown as percentage of the remaining mRNA after actinomycin on logarithmic scale. T 1/2 was determined using the means and standard deviations summarizing three independent experiments.

Article Snippet: For staining a polyclonal anti-IFIT-2 and goat-anti-rabbit IgG-PE antibody (BD Biosciences) were used.

Techniques: Expressing, Quantitative RT-PCR

Journal: BMC Immunology

Article Title: Forced IFIT-2 expression represses LPS induced TNF-alpha expression at posttranscriptional levels

doi: 10.1186/1471-2172-9-75

Figure Lengend Snippet: Impact of 3'UTRs on IFIT-2 mediated inhibition of protein expression . RAW and RAW-IFIT-2 cells were co-transfected with expression vectors encoding GFP with no 3 'UTR (GFP) or GFP with the indicated 3'UTRs (TNF, IL-6, MIP-2, VEGF, IFIT-1) and an expression vector encoding cherry with no 3'UTR for four hours. Flow cytometry of cells was performed and gated on cherry expressing cells representing efficiently transfected cells. (A) shows the mean of several experiments (n = 3–8) of the percentage of reduction of the mean GFP fluorescence of RAW-IFIT-2 compared to RAW264.7 cells for each construct used. (B) illustrates the histograms of the GFP fluorescence of one representative experiment. Asterisks indicate significant differences (p < 0.05, one-way ANOVA, Dunnett test) compared to control (GFP, no UTR).

Article Snippet: For staining a polyclonal anti-IFIT-2 and goat-anti-rabbit IgG-PE antibody (BD Biosciences) were used.

Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Flow Cytometry, Fluorescence, Construct

Journal: BMC Immunology

Article Title: Forced IFIT-2 expression represses LPS induced TNF-alpha expression at posttranscriptional levels

doi: 10.1186/1471-2172-9-75

Figure Lengend Snippet: Impact of deletions in the TNF-α 3'UTR on IFIT-2 mediated inhibition of protein expression . RAW and RAW-IFIT-2 cells were co-transfected with expression vectors encoding GFP with no 3'UTR (GFP) or GFP with the indicated 3'UTRs and an expression vector encoding cherry with no 3'UTR for four hours. (A) depicts the constructs used, (B) shows the mean of several experiments (n = 3–8) of the percentage of reduction of the mean GFP fluorescence of RAW-IFIT-2 compared to RAW264.7 cells for each construct used. Asterisks indicate significant differences (p < 0.05, one-way ANOVA, Dunnett test) compared to control (GFP, no UTR). (C) illustrates the histograms of the GFP fluorescence of one representative experiment.

Article Snippet: For staining a polyclonal anti-IFIT-2 and goat-anti-rabbit IgG-PE antibody (BD Biosciences) were used.

Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Construct, Fluorescence