Chromatin Fractionation and Western Blot Analysis

ES cells with or without 1 μM 6-TG treatment for 24 hr were harvested and total cell lysates were collected by scraping the cells from the 10 cm culture dish in 500 ul RIPA buffer with protease inhibitor cocktail (Roche). To isolate the chromatin fractions, cells were permeabilized and washed in a digitonin (Wako)-containing buffer with protease inhibitor cocktail (Roche) and then treated with the crosslinking reagent DTSSP (Pierce) on the 10-cm culture dishes as described33 (link). The total cell lysates and the chromatin fractions were then boiled in 2× Laemmli sample buffer and analyzed by SDS-PAGE and Western blotting with use of appropriate antibodies. An AlphaImager 2200 (Alpha Innotech) was used for quantitative analysis of the Western blotting results.