Quantitative deter mination of the cDNA levels was done by real time PCR using the Bio Rad iQ5 Gradient Real Time SYBR Green PCR system. Levels of cDNA were normalized to the GAPDH values of the respective samples. All results rep resent the mean SD of at least three independent experiments. Sequences of the primers http://www.selleckchem.com/products/MDV3100.html are listed in Add itional file 4 Table S1. Northern blot analysis and nuclear run on assay Additional methods for measuring the rRNA transcrip tion rate Northern blot analysis and nuclear run on assay were performed as essentially described else where, with some modifications. For the nuclear run on assay, we used Digitonin in place of NP 40 to permeabilize cells. Cells were washed twice with ice cold 1X PBS and removed from the cul ture plate using a cell scraper in 1 ml of 1X PBS per 10 cm dish and collected by centrifugation.

Cell pellets were resuspended in 1 ml of lysis buffer per 107 cells. After 10 min incubation on ice, nuclei were then collected by centri fugation and washed with lysis buffer de void of Digitonin. To perform run on reactions, aliquots of nuclei were mixed with 100 ml of 2X reac tion buffer and biotin 16 UTP in a final volume of 200 ml at 29 C for 30 min. A total of 60U of RNase free DNaseI and 6 ml of 250 mM CaCl2 were added, and the reaction mixture was incubated for an additional 10 min at 37 C. Biotinylated RNA was purified by Dynabeads M 280, a magnetic bead covalently linked to streptavidin. Dynabeads resuspended in binding buffer were mixed to an equal volume of run on RNA and subjected a 2 hr incubation at room temperature.

Beads were separated by the magnetic apparatus and washed once with 500 ml 2X SSC 15% formamide for 10 min and twice with 1 ml 2X SSC for 5 min each. Random hexamer primed cDNA was synthesized using 10 ml biotinylated RNA, and subsequently subjected to semi quantitative PCR to assay for 45 S pre rRNA transcription rate. To ensure the efficiency of the re verse transcription, the intensities of PCR products were normalized to those of GAPDH. Generation of the probe for the Northern blot analysis was based on the previous report. Chromatin immunoprecipitation and real time PCR analysis ChIP assay was performed essentially as described previ ously. Crosslinked, sonicated chromatin was pre cleared before being incubated with 2. 5 ug of the indicated antibodies and rotated at 4 C overnight.

Nor mal mouse or rabbit IgG was used for the mock immunoprecipitation. After extensive washes, immunocomplexes were treated with Proteinase K and decrosslinked. Bound DNA in the precipitates, as well as input DNA, was extracted, purified, and subjected Dacomitinib to real time PCR analysis using primers corresponding to different regions of the rDNA repeat unit. Real time PCR reactions were conducted on the Bio Rad iQ5 Gradient Real Time PCR system, using the 2X SYBR Green Master mix. Results were corrected for nonspecific binding to IgG and pre sented as percentage of input DNA.