2% Triton X 100 in PBS for 10 min, followed by 3 washes in PBS. Blocking was carried out for 30 min at room temperature with 5% normal goat serum in PBS. Cells were incubated www.selleckchem.com/products/MLN-2238.html with mouse anti H2A. X for 1 hr, followed by 3 PBS washes. Secondary antibody, anti mouse Alexa Fluor 488, was applied for 1 hr, fol lowed by 3 washes in PBS. Following a rinse with ddH2O, coverslips were mounted on glass slides using Vectashield mounting medium with DAPI. Fluorescence was assessed using the Axioskop 2 MOT microscope. Flow Cytometric Analysis of g H2A. X Expression Following treatment, cells were trypsinized, washed in PBS and fixed on ice with 1% paraformaldehyde for 15 min. After centrifugation, the cell pellet was resus pended in 500 ul of PBS and transferred to a tube con taining 4.

5 ml of cold 70% ethanol and kept at 20 C for a minimum of 2 hrs. Cells were centrifuged and then washed twice in BSA T PBS. Following the sec ond wash, the cell pellet was resuspended in BSA T PBS containing mouse anti gamma H2A. X primary antibody at 1 100 and incubated overnight at 4 C. Cells were then washed once in BSA T PBS and resuspended in BSA T PBS containing anti mouse Alexa Fluor 488 secondary antibody at 1 400 and incubated at room temperature in the dark for 1 hr. Cells were washed once in BSA T PBS and resuspended in PBS containing 50 ug ml propidium iodide and 5 ug ml RNAse A. Cells were analyzed on a Coulter Epics XL flow cytometer and the resulting data was assessed using ModFit software. Chromatin Immunoprecipitation Assay Cells were fixed in 1% formaldehyde for 20 min at room temperature.

Fixation was stopped by quenching with 2. 5 mM glycine solution to a final concentration of 200 mM for 5 min. Cells were then washed twice with ice cold PBS and harvested in 1 ml cold PBS by centrifugation for 5 min at 5,000 rpm. The pellet was resuspended in 90 ul lysis buffer supplemented with 1X Protease Inhibitor Cocktail, 1 mM 1,4 dithio DL threitol, and 1 mM phenylmethylsulfonyl fluoride. The lysates were sonicated using a Sonicator 3000 to shear DNA to an average size of 300 to 1000 base pairs and then cleared of debris by centrifugation at 14,000 rpm for 15 min. Input controls were removed from each sample and stored at 20 C. The sonicated lysates were diluted 10 fold with dilu tion buffer, supplemented with 1X Protease Inhibitor Cocktail, 1 mM DTT and 1 mM PMSF, and immunoprecipitated by overnight rota tion at 4 C with rabbit anti acetyl H4 primary antibody.

Negative controls were incubated in the absence of primary antibody. Immune complexes were collected by 2 hr rotation at 4 C with the addi tion of 40 ul of protein A agarose salmon sperm DNA 50% slurry to both positive samples and negative controls. The beads were pelleted gently by centrifugation for 1 min at 3,000 rpm at 4 C and washed Carfilzomib with 1 ml of the following buffers by rotation for 10 min at 4 C Buffer A once, Buffer B once, Buffer C once and TE washing buffer twice.