Aprotinin protease inhibitors

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.

Caki-2 cells (400,000 cells) were seeded in 6 cm dishes plates and under 2 μg/ml doxycycline for 3 days. Then cells were treated with 100 μg/ml cycloheximide (Selleck) for 0, 2, 6, 10, or 24 h. At each time point, cells were harvested and lysed in radioimmunoprecipitation assay buffer with 1:1000 leupeptin, pepstatin A, and aprotinin protease inhibitors (Cayman Chemical).