Phosphatase inhibitor cocktail 1 and 2

Chromatin fractionation was performed as described [70 (link)]. In brief, THP-1 cells (1 × 10⁵ cells/well) were treated with the vehicle (DMSO) or with 10 μM DT for 24 hours, the conditioned medium was collected. Then, the medium of DU 145 cells was replaced with the conditioned medium or regular medium and subsequently cultured for 24 hours. After the DU145 cells were treated with indicated conditioned medium for 24 hours, the cells were washed twice by cold-PBS. Cell pellets were resuspended in buffer A (50 mM Hepes, pH 7.9, 10 mM potassium chloride (KCl), 1.5 mM MgCl₂, 0.34M Sucrose, 10% Glycerol (v/v), 1 mM dithiothreitol (DTT), protease inhibitor cocktail (Roche), 0.1% Triton X-100 (v/v) and phosphatase inhibitor cocktail I and II (Sigma) on ice. After centrifuge, pellets were lysed by buffer B (3 mM EDTA, 0.2 mM ethylene glycol tetraacetic acid (EGTA), 1 mM DTT, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail I and II (Sigma) After centrifuge, pellets were washed twice by washing buffer I (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, 150 mM NaCl, protease inhibitor cocktail (Roche) and buffer II (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, 250 mM NaCl, protease inhibitor cocktail (Roche). After washing, pellets were sonicated and lysed by E1A lysis buffer. Then proteins were analyzed by Western's blot analysis.

Total proteins from BMDM or iM were extracted using lysis buffer (50 mM Tris pH8, 300 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 1 mM EDTA, phosphatase inhibitor cocktail-1 and -2 (Sigma Aldrich) and complete protease inhibitor cocktail (Roche)). Proteins were separated on 3–8% Tris-Acetate gel or 4–12% Bis Tris gel (Life Technologies), transferred on a nitrocellulose membrane (Amersham) and blotted with the indicated antibodies. Following staining with HRP-coupled secondary antibodies, the proteins were detected with ECL Prime by chemiluminescence (Amersham).

Whole cell lysates were prepared by lysing PBS-washed OSU-CLL cell pellets in cold lysis buffer containing phosphatase inhibitor cocktail 1 and 2, protease inhibitor cocktail P8340 and 1mM phenylmethylsulfonyl fluoride (all from Sigma). Protein was quantified by the BCA method (Pierce). Protein (25ug/lane) was separated on 12% polyacrylamide gels and transferred onto nitrocellulose. After antibody incubations, proteins were detected with chemiluminescent substrate (Advansta) and quantified using a ChemiDoc system with Quantity One software (Bio-Rad Laboratories). The following antibodies were used for detection, anti-ACTB (Santa Cruz Biotechnologies) and anti-γH2AX (Abcam).