Anti ncaph

Flag-tagged full-length NCAPH and GEN1 proteins were expressed in HEK293 cells and purified with FLAG M2 monoclonal antibody affinity gel (Sigma) according to manufacturer’s instructions. GST-tagged full-length GEN1 protein was expressed in HEK293 cells and purified with Glutathione-Sepharose 4B (Thermo Fisher Scientific) according to manufacturer's instructions. HEK293 cells transfected with three truncation domain constructs Flag-tagged-NCAPH (N-NCAPH [1–250], M-NCAPH [251–500], and C-NCAPH [501-741]) or control vector and then purified with Glutathione-Sepharose 4B or FLAG M2 monoclonal antibody affinity gel (Sigma). The primary antibodies were acquired from commercial sours: anti-Flag (Sigma), anti-GST (Cell Signaling Technology), anti-NCAPH (Novus Biological), anti-NCAPD2 (Bethyl Laboratories), anti-GAPDH (Thermo Fisher Scientific), anti-LMNA (Bethyl Laboratories). The ATM-kinase inhibitor (KU-55933) was acquired from Sigma.

Cells were grown on Thermo Fisher Scientific Nunc Lab-Tek II Chamber Slides & trade, fixed with 4% paraformaldehyde for 15 min or PTEM buffer containing 20 mM PIPES (pH 6.8), 0.2%. Triton X-100, 10 mM EGTA, and 4% paraformaldehyde for 10 min, and permeabilized with 0.5% Triton X-100 for 5 min. The fixed cells were incubated for 1 h with % bovine serum albumin and then incubated overnight at 4°C with primary antibody, then the next day add the fluorophore-conjugated secondary antibody for 2 h. Samples were mounted in Vectashield Mounting Medium with DAPI (Vector laboratories, USA), and the results were observed under a confocal microscope (Zeiss LSM 710 or LSM 800, software ZEN; Zeiss, Germany). The primary antibodies were acquired from commercial sours: anti-NCAPH (Novus Biological), anti-GEN1 (Novus Biological), anti-p-H2AX (Millipore), anti-CREST (ImmunoVision, USA), anti-GFP (Invitrogen), and anti-alpha Tubulin (Abcam).