2 metcaptoethanol

4x10⁶ eHAP cells were seeded per 10cm dish 24 h prior to collection. Cells were treated with 10uM formyl-dU or DMSO control for 1 h prior to collection. Cells were scraped in 1ml ice-cold PBS. 50% of the sample was kept on ice for whole cell control. The remaining cells were spun down for 4 min at 500 g and resuspended in 200ul CSK buffer (10mM PIPES pH7.0, 100mM NaCl, 300mM Sucrose, 1.5mM MgCL₂, 5mM EDTA, 0.5% Triton 1x phosphatase (Phos-Stop, Roche) and protease (Complete, EDTA-free, Roche) inhibitor mixes)) and incubated on ice for 10 min. Cells were spun down at full speed for 10 s 150ul of supernatant (soluble fraction) was collected. Residual soluble fraction was removed and the chromatin pellet was washed in 500ul of CSK. Whole cell chromatin pellets were resuspended in 200ul 1X NuPAGE LDS sample buffer (Invitrogen) and 1% 2-metcaptoethanol (Sigma-Aldrich). 50uL of 4x NuPAGE LDS sample buffer (Invitrogen) and 4% 2-metcaptoethanol (Sigma-Aldrich) was added to soluble fraction. Samples were sonicated with a probe at medium intensity for 10 s in a Soniprep 150 instrument and then incubated at 95°C for 10 min. 20ul of each fraction was loaded and subjected to SDS-PAGE as above.

For whole cell lysates, PBS washed cells were lysed in RIPA Buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1x phosphatase (Phos-Stop, Roche) and protease (Complete, EDTA-free, Roche) inhibitor mixes) on ice for 20 min. Lysates were sonicated with a probe at medium intensity for 5 s in a Soniprep 150 instrument and clarified by centrifugation at 13000 g for 15 min at 4°C. Protein concentration was quantified using the DC Protein Assay (Bio-Rad) according to the manufacturer’s instructions. Proteins were denatured in 2X NuPAGE LDS sample buffer (Invitrogen) and 1% 2-metcaptoethanol (Sigma-Aldrich) for 5 min at 95°C. Proteins were separated by SDS-PAGE using NuPAGE mini gels (Invitrogen) and transferred onto 0.2 μm pore Nitrocellulose membrane (Amersham Protran; Sigma-Aldrich). Membranes were blocked with 5% skim milk/TBST (TBS/0.1%Tween-20) for 1 h at room temperature and probed with the indicated primary antibodies overnight at 4°C. Membranes were then washed 3 times for 10 min with TBST, incubated with appropriate secondary antibodies conjugated to a horseradish peroxidase (HRP) for 1 h at room temperature and washed again 3 times for 10 min with TBST. Immunoblots were developed using Clarity or Clarity Max Western ECL Substrate (Bio-Rad).