Nuclear extracts (NE) were prepared as described (9 (link)). For IPs from JSL1 cells, 100 μg of NE, pretreated with RNase A and RNase T1, were incubated with 5 μg PSF antibody (Sigma) or GST antibody (GE Healthcare) in 400 uL IP buffer with rotation (20 mM Tris pH 7.5, 100 mM NaCl, 0.2 mM EDTA, protease inhibitor) overnight at 4°C. Extracts were then incubated for 1 h with protein G Dynabeads (Life Technologies) with rotation. Beads were washed three times with 400 μl IP buffer supplemented with 200 mM NaCl and eluted using 2x Laemmli buffer. Inputs and eluted proteins were then analyzed by western blot. For co-IPs of FLAG-tagged PSF, the above procedure was repeated with NE from JSL1 cells stably expressing FLAG-PSF WT or mutants, with anti-FLAG antibody (Sigma) used to precipitate protein complexes.
For pulldown assays, 40 μg GST-tagged protein was bound to glutathione sepharose 4B beads previously equilibrated and resuspended in 1 ml of PBS for 1.5 h at 4°C with rotation. Bound proteins were washed twice with PBS. Prey proteins were incubated with immobilized bait for 1.5 h at 4°C in 100 μl PBS with rotation. Bound complexes were washed three times with PBS supplemented with 300 mM NaCl before elution in 2x Laemmli buffer. Inputs and eluted proteins were then analyzed by western blot.
For pulldown assays, 40 μg GST-tagged protein was bound to glutathione sepharose 4B beads previously equilibrated and resuspended in 1 ml of PBS for 1.5 h at 4°C with rotation. Bound proteins were washed twice with PBS. Prey proteins were incubated with immobilized bait for 1.5 h at 4°C in 100 μl PBS with rotation. Bound complexes were washed three times with PBS supplemented with 300 mM NaCl before elution in 2x Laemmli buffer. Inputs and eluted proteins were then analyzed by western blot.