40 mg/mL RNase A and 20 mg/mL TPCA-1 concentration proteinase K, 10 mM EDTA and 40 mM Tris-HCl pH 6.5 were added and samples were then incubated 2 hours at 45°C. Samples were then extracted in phenol-chloroform-isoamylic acid (25:24:1), ethanol-precipitated and finally centrifuged at 13000 rpm for 45 minutes at 4°C. Pellets were washed with 70% ethanol, centrifuged at 8000 rpm for 5 minutes at 4°C and finally resuspended in 60 μL of H2O. 2 μL of each sample were used as template selleck for subsequent PCR analysis and 32 amplification cycles were used. Amplification of the IL-8 promoter fragment, using SYBR®Green Taq, was performed using the primers: pIL-8F
(forward) 5′- CAGAGACAGCAGAGCACAC-3′ and pIL-8R (reverse) 5′-ACGGCCAGCTTGGAAGTC-3′ amplifying a 101 bp fragment. All PCR signals from immunoprecipitated DNA were normalized to PCR signals from non-immunoprecipitated input DNA. The signals obtained by precipitation with the control IgG were subtracted from the signals obtained with the specific antibodies. Results are expressed as percentage of the input: signals obtained from the ChIPs were divided by signals obtained from an input sample; this input sample represents the amount of chromatin used in the ChIP. Calculations take into account the values of at least three independent experiments. Statistical Analysis Statistical significance between groups was assessed by Student’s t test. Data are expressed as means ±
standard deviation (SD). All experiments were repeated https://www.selleckchem.com/products/verubecestat-mk-8931.html at least three times. A p value < 0.05 was considered to be statistically significant. Acknowledgements This work was supported by grant from MIUR (PRIN07) to LC. References 1. Hamon MA, Cossart P: Histone modifications and chromatin remodeling during bacterial infections. Cell Host Microbe 2008, 4:100–109.PubMedCrossRef 2. Minárovits J: Microbe-induced epigenetic alterations in host cells: the coming era of patho-epigenetics of microbial
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