A recent study identified numerous long ncRNAs transcribed from active promoter

Normal human ventricular tissues were from four healthy male individuals involved ABT-737 852808-04-9 in road traf fic accidents. At the time of trans plantation or donor harvest, whole hearts were removed after preservation and transported in cold cardioplegic solution similar to the procedure described before at Imperial College, London. Following analysis by a cardiovas cular pathologist, left ventricular segments were cut and stored immediately in RNAlater. Individual patient details are listed in Additional file 1. Immunoblot analysis Whole cell lysates were prepared by scraping cells into a lysis buffer. To prepare nuclear lysates, cell membranes cytoplasms of harvested cells were lysed in an ice cold cytoplasmic lysis buffer and the nuclei were washed with the same buffer before being lysed with a buffer containing 0.<br><br> 45 M NaCl, 10 mM HEPES pH7. 4 and 1x Roche Com plete Mini Protease Inhibitor Cocktail. Protein concen tration was determined using a BCA Protein AEB071 Sotrastaurin Assay Kit. Equal pro tein amounts were resolved by SDS PAGE, transferred to a polyvinylidene fluoride membrane and incubated with primary antibodies against the following proteins, p53, NF κB, STAT3, SF2 and RhoGDI. Goat anti mouse and anti rabbit were used as secondary antibodies. The membrane was incubated with SuperSignal West Pico Chemiluminescent Substrate, SuperSignal West Dura Extended Duration Substrate, SuperSignal West Femto Maximum Sensitivity Chemiluminescent Substrate or ECL Advance Western Blotting Detection Kit before being exposed to an X ray film.<br><br> AG-014699 Immunoprecipitation Cells were scraped into the lysis buffer and protein con centration was determined as described above. Protein A beads were prewashed twice with phosphate buffered saline and suspended in the lysis buffer. Total protein lysate was incubated with 30 ul of the protein A beads for an hour at 4 C and centrifuged. The supernatant was then removed to a fresh tube and incu bated overnight at 4 C with primary antibodies and IgG as indicated. The next day, 30 ul of the protein A beads were added and incubated for an hour at 4 C. Following the incubation, beads were washed three times with phos phate buffered saline. Proteins were eluted with SDS loading buffer, resolved by SDS PAGE, and transferred to a membrane for immunoblot analysis as described above.<br><br> Chromatin immunoprecipitation and sequential ChIP Chromatin immunoprecipitation was performed using a ChIP Assay Kit, primary antibodies raised against p53, DO 1, sc 126 Santa Cruz for human NF κB and histone H3, and IgG. For tissue ChIP, the heart tissues were finely chopped, cross linked and homogenized prior to the procedure. Cross linked chromatin was fragmen tized to 1 kb by sonication. Regions of interest were amplified from the immunoprecipitated DNA by qPCR using SYBR GreenER qPCR SuperMix Universal. The primers for the rat 32280 7 site were 5 TAG PCR signals were standardized with signals from amplification of 18s rRNA genes with the same primers probe and protocol as described above. For sequential ChIP assays, complexes from the primary ChIP were eluted twice with 10 mM dithiothreitol for 20 minutes at 37 C, diluted 10 times with re ChIP buffer followed by re immunoprecipitation with the indicated second pri mary antibody, and then again subjected to the ChIP pro cedure.