Western blot and co-immunoprecipitation (CoIP) experiments

Cells were collected from plates by scraping in ice-cold phosphate-buffered saline (PBS) with PMSF and aprotinin, pelleted, and flash-frozen in liquid nitrogen.

For western blot analysis, cell pellets where thawed on ice, resuspended to 1 mL/10 cm plate of low-salt lysis buffer (0.1 M NaCl, 25 mM HEPES pH 7.5, 1 mM MgCl2, 0.2 mM EDTA, 0.5% NP-40 and protease inhibitors), with 125 U/mL of benzonase (Novagen, EMD Millipore), passed through a 25G needle, rocked at 4°C for 1 hr and 5M NaCl was added to reach a final concentration of 0.2 M. Lysates were then rocked at 4C for 30 min and centrifuged at maximum speed at 4°C. Supernatants were quantified by Bradford. 15 μg of proteins were loaded on 8% Bis-Tris SDS-PAGE gel and transferred onto nitrocellulose membrane (Amersham Protran 0.45 um NC, GE Healthcare) for 2 hr at 100 V.

For chemiluminescent western blot detection with HRP-conjugated secondary antibodies, after the transfer the membrane was blocked in TBS-Tween with 10% milk for 1 hr at room temperature and blotted overnight at 4°C with primary antibodies in TBS-T with 5% milk. HRP-conjugated secondary antibodies were diluted 1:5000 in TBS-T with 5% milk and incubated at room temperature for an hour.

For fluorescence detection, after the transfer the membrane was blocked with the Odyssey Blocking Buffer (PBS) for 1 hr at room temperature, followed by overnight incubation at 4°C with primary antibodies in Odyssey Blocking Buffer (PBS) and PBS (1:1). IRDye secondary antibodies were used for detection at 1:5000 dilution and 1 hr incubation at room temperature. After extensive washes, the membrane was scanned with a LI-COR Odyssey CLx scanner.

For co-immunoprecipitation experiments (CoIP), cell pellets where thawed on ice, resuspended to 1 ml/10 cm plate of cell lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40 and protease inhibitors), and incubated on ice for 10 min. Nuclei were pelleted in a tabletop centrifuge at 4°C, at 4000 rpm for 10 min, and resuspended to 0.5 ml/10 cm plate of low salt lysis buffer either with or without benzonase (600 U/ml) and rocked for 4 hr at 4°C. After the 4-hr-incubation, the salt concentration was adjusted to 0.2M NaCl final and the lysates were incubated for another 30 min at 4°C. Lysates were then cleared by centrifugation at maximum speed at 4°C and the supernatants quantified by Bradford. In a typical CoIP experiment, 1 mg of proteins was diluted in 1 ml of CoIP buffer (0.2 M NaCl, 25 mM HEPES pH 7.5, 1 mM MgCl₂, 0.2 mM EDTA, 0.5% NP-40 and protease inhibitors) and pre-cleared for 2 hr at 4°C with protein-G sepharose beads (GE Healthcare Life Sciences) before overnight immunoprecipitation with 4 mg of either normal serum IgGs or specific antibodies as listed above. Some pre-cleared lysate was kept at 4°C overnight as input. Protein-G-sepharose beads precleared overnight in CoIP buffer with 0.5% BSA were then added to the samples and incubated at 4°C for 2 hr. Beads were pelleted and all the CoIP supernatant was removed and saved for phenol-chloroform extraction of DNA. The beads were then washed extensively with CoIP buffer, and the proteins were eluted from the beads by boiling for 5 min in 2X SDS-loading buffer and analyzed by SDS-PAGE and western blot.