Histone h3 d1h2 xp

tsA201 cells transfected with recombinant hGR isoforms were either lysed in ice-cold lysis buffer (Agilent) supplemented with cOmplete Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and supernatants harvested and normalized, or were fractionated into nuclear, cytoplasmic, and membrane extracts using a cell fractionation kit (Cell Signaling Technology, Danvers, MA). Proteins were run on a 4–20% BioRad Criterion TGX gel (Hercules, CA), then transferred to a polyvinylidene difluoride membrane (BioRad). Membranes were blocked with 5% nonfat dry milk, washed, and incubated overnight with Glucocorticoid Receptor (D8H2) XP (1:1000), MEK1/2 (D1A5) (1:1000), AIF (D39D2) XP (1:1000), or Histone H3 (D1H2) XP (1:2000) rabbit monoclonal antibodies (Cell Signaling Technology) in 5% nonfat milk or bovine serum albumin. A secondary anti-rabbit-HRP (GE Healthcare, Piscataway, NJ) in 5% nonfat milk at 1:2000 was used for protein visualization via chemiluminescence using the ECL Prime Western Blot Detection System (GE Healthcare).

Cells were fixed with 4% paraformaldehyde for 15 min, followed by 30 min of permeabilization with 0.1% Triton X-100. A 5% BSA solution in PBS was used for blocking for an hour before the cells were incubated overnight with primary antibodies at 4°C (anti-RNA polymerase II CTD repeat YSPTSPS [phospho S5], abcam ab513 at 1:1000 dilution, anti-phospho-histone H2AX [Ser139; anti-γH2AX], clone JBW301, Merck 05-636 at 1:1000 dilution, anti-Chk1 [phospho S345] antibody, abcam ab47318 at 1:500 dilution, anti-HP1α antibody, clone15.19s2, Merck 05-689 at 1:1000 dilution, anti-trimethyl-histone H3 [Lys9; D4W1U], Cell Signalling Technology 13969 at 1:800, anti-ATM [phospho S1981], abcam ab36810 at 1:500 dilution, Histone H3 [D1H2] XP, Cell Signalling Technology 4499 at 1:500 dilution). Fluorescent-tagged secondary antibodies (Alexa Fluor 546 goat anti-mouse/rabbit [A11030/A11010] and Alexa Fluor 647 goat anti-mouse/rabbit [A21236/A21245]) were incubated at room temperature for 1 h before imaging.