The threshold cycle num bers for each PCR products were Lenalidomide determined, and the relative expression levels for all genes were obtained using the 2 Ct calculation. To measure the transcript levels of four composite Zea mays plasma membrane H ATPases, we used a degenerated primer pair for the ZmMHA gene de signed by Geilfus et al. Zea mays xyloglucan endo transglucosylase homolog 1 was used as a representative to study xyloglucan endotransglucosylase. Quantitative RT PCR primers were designed by Primer BLAST to amplify about 200 bp fragments. Preliminary experiments were done to ensure the amplifi cation of a single PCR product for the analyzed gene and standard curves were generated for each primer set to de termine their efficiency. The sequences and the PCR effi ciencies of the primers used for quantitative RT PCR are listed in Table 1.
In the preliminary experiment, Inhibitors,Modulators,Libraries we also tested the expression of three common reference genes such as Actin, 18S rRNA and UBQ, in the control and 200 mM NaCl treated seedlings, and we observed that actin transcription was the most stable as the CT value of actin did not vary much at the same content of DNA template between the control and treated groups. The beta actin gene was used as a reference gene, also because some publica tions reported that its expression in maize roots is litt le affected by salt stress. RT PCR was repeated three times for each sample from three independent experiments. Chromatin immunoprecipitation ChIP assays were performed using standard procedures.
Six day old maize seedlings were further grown in 1/2 Hoaglands nutrient solution with or without 200 mM NaCl for 48 h and 20 g fresh samples were ground to powder in liquid N2, suspended in TBS buffer, then filtered, washed twice by different concentration of sucrose Inhibitors,Modulators,Libraries solution, and centrifuged. Equal amounts of chromatin extract was digested into 200 500 Inhibitors,Modulators,Libraries bp with micrococcal nuclease at 37 C for 10 min. Chromatin was precleared with protein A sepharose at 4 C for 3 h and then Inhibitors,Modulators,Libraries incu bated over night at 4 C with 10 ul anti H3K9Ac and 10 ul rabbit serum. After immunoprecipitation, the extracts were gradient eluted by different concentrations of NaCl solutions, once with a low salt buffer, then once with a middle salt buffer, and finally once with a high salt buffer, and centrifuged.
Subsequently, the precipitations were eluted twice with an elution buffer at 65 C for 15 min, and centrifuged to collect the supernatant. Next, the DNA in Inhibitors,Modulators,Libraries the supernatant was extracted with a standard procedure to perform quantitative real time polymerase chain reac tion with the SYBR Green Real time PCR Master add to your list Mix. The beta actin gene as a con trol gene was used for normalization of ChIP QPCR, which can be reliably used with high quality measurements. The extract precipitated with rabbit serum was used as a negative control.