Ab177309

Cells were fixed in 100% ice-cold methanol at −20 °C for 10 min, washed three times with 1X PBS and further permeabilized in 0.3% Triton X-100 for 10 min. Following blocking in 10% normal goat serum (MP Biomedicals), cells were incubated with the primary antibody in blocking buffer at 4 °C overnight. The following antibodies were used for immunofluorescence: H4/H2AS1ph (ab177309, Abcam; 1:2000), Lamin A/C (ab238303, Abcam; 1:1000), H3K4me3 (ab8580, Abcam; 1 μg/ml) and H3K36me3 (ab9050, Abcam; 1 μg/ml). Next, cells were washed three times with 1X PBS and following incubation with Alexa Fluor 568 goat anti-rabbit (A11011, Thermo Fisher Scientific; 1:1000) and Alexa Fluor 488 goat anti-mouse (A11001, Thermo Fisher Scientific; 1:1000) secondary antibodies diluted in 10% normal goat serum for 1 h at room temperature, nuclei were stained with DAPI (Dako) or Hoechst 33342 (Invitrogen). Samples were imaged on a ZeissAxio Observer.A1 microscope. For confocal and super resolution microscopy imaging was carried out on a ZEISS LSM 900 with Airyscan 2 using Zen blue for acquisition and processing. Airyscan2 images were processed using the default deconvolution settings and histogram stretching, applied when required, was identical between control and treated samples for each channel.

Ten million cells were harvested in 1X PBS and lysed in 1 ml ice-cold IP buffer (20 mM Tris-HCL p^H8, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA and 1X protease inhibitor cocktail) for 30 min with constant mixing at 4 °C. Following centrifugation at 12,000 rpm for 20 min at 4 °C, the soluble supernatant fraction was pre-cleared with Protein A sepharose beads for 1 h at 4 °C. Five percent of the lysate was kept as “Input” to serve as a positive control. Lysates were then mixed with 60 μl of Protein A sepharose beads that were pre-incubated with 4 μg of H4/H2AS1ph (ab177309, Abcam), Lamin A/C (sc-7292 X, Santa Cruz) or IgG (Biogenesis 5180-2104) antibodies for 4 h and blocked in salmon sperm DNA for 40 min. Following overnight incubation with constant agitation at 4 °C, the antibody-beads-protein complexes were centrifuged and washed three times with low salt buffer (10 mM Tris-HCL p^H7.4, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100 and 1X protease inhibitor cocktail) and IP samples were eluted in 2X Laemmli buffer at 95 °C for 10 min.