Journal: Fems Microbiology Letters

Article Title: Regulation of Cu(I)/Ag(I) efflux genes in Escherichia coli by the sensor kinase CusS

doi: 10.1111/j.1574-6968.2012.02529.x

Figure Lengend Snippet: Data shows the relative expression from the cusC gene +/− SEM, as determined in E. coli wild-type (gray), E. coli ΔcusS (checkered) and E. coli ΔcusS/pBADcusS (diagonal lines) after exposure to 5 µM AgNO3 for 0, 2 and 4 hours. Total RNA from cells was extracted, reverse transcribed and cDNA was subjected to real-time PCR using primers specific for cusC. ANOVA analysis with multiple comparisons showed that transcription from cusC is absent when cusS is disrupted (P < 0.0001).

Article Snippet: First strand cDNA was prepared from 2 µg of total RNA using the Superscript III cDNA synthesis kit (Quanta Biosciences).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal:

Article Title: Egr-1 Regulates Expression of the Glial Scar Component Phosphacan in Astrocytes after Experimental Stroke

doi: 10.2353/ajpath.2008.070648

Figure Lengend Snippet: Egr-1 binds to the phosphacan promoter in vivo. A: HeLa cells were transiently transfected with Egr-1 full-length human cDNA and subjected to ChIP with anti-Egr-1 antibody or rabbit IgG isotype control. Specific primers were used to amplify the area containing the putative Egr-1 site (lanes 1), or a distal, 4.5-kb upstream fragment (lanes 2). Genomic DNA input shows that the primers amplify bands of the expected size. Immunoprecipitation of sheared chromatin (ChIP) with anti-Egr-1 (α-Egr-1) produces a band of stronger intensity as compared with the distal site product and to control isotype IgG. B: HeLa cells were stimulated for 2 hours with PMA and ionomycin to induce endogenous Egr-1 and subjected to ChIP with anti-Egr-1 antibody or rabbit IgG isotype control. Precipitated DNA fragments were amplified with the same primers as in A surrounding the Egr-1 site (lanes 1) or the distal area (lanes 2). Genomic DNA input shows that primers amplify bands of the expected size. Immunoprecipitation of sheared chromatin (ChIP) with anti-Egr-1 (α-Egr-1) produces a band of stronger intensity as compared with the distal site product and to control isotype IgG. C: Immunoprecipitated DNA from the analysis in B was amplified and quantified by real-time PCR using primers surrounding the Egr-1 binding site or the distal area. Fold differences in the amounts of PCR products obtained with anti-Egr-1 antibody relative to isotype controls were calculated as described in Materials and Methods. Average values with SEM from five independent experiments are depicted. There is ∼6- to 10-fold higher amplification of the Egr-1 site fragment compared with the control fragment from the distal area.

Article Snippet: The three fragments were amplified using human genomic DNA as template (prepared with the Wizard genomic DNA purification kit, Promega) and inserted into the pGL3 promoter vector (Promega).

Techniques: In Vivo, Transfection, Control, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Binding Assay