Trap150

Cells grown on coverslips were washed once with 1× PBS and fixed with 2% formaldehyde (in 1× PBS) for 15 min followed by three washes with 1× PBS for 5 min each. The cells were then permeabilized for 5 min with 0.2% TritonX-100 and washed three times with 0.5% normal goat serum (NGS, Gibco-Thermo Scientific, Waltham, MA, USA) in 1× PBS. The coverslips were then incubated with primary antibodies (Btf (WU10; 1:2500), TRAP150 (1:1000, Bethyl catalog number A300-956A)) diluted in 0.5% NGS-1 × PBS cell side up in a humidified chamber for one hour at room temperature. After three washes with phosphate buffered saline with 0.5% normal goat serum (PBS/0.5% NGS), the cells were incubated in appropriate secondary antibodies conjugated with Texas Red, Cy5, or FITC (1:500; Jackson Immunoresearch Laboratories, West Grove, PA, USA). DNA was stained with DAPI (10 μg/mL). Finally, the coverslips were mounted in anti-fade polyphenylenediamine medium and sealed with a clear nail polish. Cells were imaged using a DeltaVision RT microscope equipped with a 60x objective (1.4 numerical aperture; Olympus, Tokyo, Japan), and softWoRx 2.50 software (DeltaVision by Applied Precision/GE Healthcare, Issaquah, WA, USA). Raw images were displayed as volume projections. Mitotic stage was scored according to the distinctive appearance of chromatin at prophase, metaphase, anaphase, and telophase.

The following antibodies were used throughout as noted: PSF (Sigma P2860 for IP, Abnova H00006421-A01 for WB), TRAP150 (A300–956A, Bethyl Laboratories), FLAG (2368, Cell Signaling), GST (27–4577–01, GE Healthcare), His (AM1010a, Abgent), hnRNP L (4D11, Abcam), p54^nrb/NONO (MA3–2024, Affinity Bioreagents), MATR3 (NB100–1761, Novus Biologicals), PSPC1 (a gift from Dr. Archa Fox).