4 to 12 sds polyacrylamide gel

Immunoblotting was performed as previously described (12 (link)). Protein samples from cell lysates, coimmunoprecipitations, cycloheximide chase assays, and peptide pulldowns were quantified by BCA assays (78 (link)). After sample concentration was normalized, samples were denatured for 10 min at 95 °C, separated on a 4 to 12% SDS-polyacrylamide gel (Invitrogen), and transferred to a nitrocellulose membrane (Millipore). The membrane was blocked with 5% w/v bovine serum albumin (VWR International) in PBS containing 0.1% v/v Tween 20 for 1 h at room temperature and then incubated in a 1:1000 dilution of primary antibodies overnight at 4 °C. The primary antibodies were detected by incubating the membranes in a 1:10,000 dilution of goat-anti-rabbit or goat-anti-mouse IgG secondary antibodies (LI-COR Biotechnology) conjugated to IRDye 800CW (lot no. D20510-25) or IRDye 680 (lot no. D20920-25), respectively, for 1 h at room temperature. The signals were visualized using an Odyssey Clx imager (LI-COR Biotechnology). Any quantification was performed by band densitometry, normalizing to β-actin for the cycloheximide chase assays and to 10% input for the peptide pulldowns.

Peptide pulldowns were performed as previously described (12 (link)). Streptavidin Agarose Ultra Performance resin (15 μl) (Solulink) was washed three times with binding buffer (50 mM Tris pH 7.8, 150 mM NaCl, 0.5 mM DTT). The resin was resuspended in 300 μl of binding buffer with 2 μg of biotin-labeled peptide (AnaSpec) plus 1:1000 leupeptin, pepstatin A, and aprotinin protease inhibitors (Cayman Chemical), and samples were rotated at 4 °C for 1 h. The following peptides were used: H3(1–30), H3K14ac(1–30), and H3K14/18/23/27ac(1–30). Caki-2 cells (5 × 10⁶) were harvested and lysed in 2 ml of buffer A (20 mM Hepes pH 7.9, 25 mM KCl, 0.1% v/v Nonidet P-40, 10% v/v glycerol, plus protease inhibitors) and centrifuged. The nuclei were then resuspended in 250 μl of IP Buffer (25 mM Tris pH 8, 300 mM NaCl, 1% v/v Nonidet P-40, 1 mM EDTA, plus protease inhibitors) and rotated at 4 °C for 10 min. The samples were then spun down at 15,000 rpm for 15 min. The 250 μl of lysate was added to the peptide and resin solution and rotated overnight. The samples were washed for 10 min three times in binding buffer. The resin was resuspended in 1 × Bolt lithium dodecyl sulfate sample buffer (Invitrogen) and boiled for 5 min. Nuclear lysate input and the samples were loaded onto a 4 to 12% SDS-polyacrylamide gel (Invitrogen) for immunoblotting.

Immunoprecipitation was performed as previously described (64 (link)). Caki-2 cells (1 × 10⁷) were harvested and lysed in 2 ml of buffer A (20 mM Hepes pH 7.9, 25 mM KCl, 0.1% v/v Nonidet P-40, 10% v/v glycerol) plus 1:1000 leupeptin, pepstatin A, and aprotinin protease inhibitors (Cayman Chemical) and centrifuged at 600g for 10 min. The nuclei were then resuspended in 250 μl of immunoprecipitation (IP) buffer (25 mM Tris pH 8.0, 300 mM NaCl, 1% v/v Nonidet P-40, 1 mM EDTA, plus protease inhibitors) and rotated at 4 °C for 30 min. The extracts were cleared by centrifugation at 21,000g for 30 min. The cleared extract was precleared with normal immunoglobulin G (IgG)-conjugated protein A/G magnetic beads (Pierce) for 20 min. One microgram of specific IgG was used per 0.2 mg lysate for immunoprecipitation. After overnight incubation, immunocomplexes were captured using protein A/G magnetic beads following a 2-h incubation. The beads were washed twice in chromatin IP buffer and three times in high stringency wash buffer (20 mM Hepes pH 7.9, 500 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM EDTA). The proteins were eluted in 1× lithium dodecyl sulfate loading dye (Thermo Fisher Scientific) by heating at 70 °C for 10 min. Samples were heated at 95 °C for 10 min then loaded onto a 4 to 12% SDS-polyacrylamide gel (Invitrogen) for immunoblotting.