Affinity Purification-Mass Spectrometry of NUSAP1 Interactors

Affinity purification–mass spectrometry (AP–MS) identification of binding partners was performed as described previously [69 (link)]. Notably, 293T cells transfected with Flag-NUSAP1 were lysed in 0.2% NP-40 buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM CaCl₂, 1 mM EDTA, 0.2% NP-40, Halt protease, and phosphatase inhibitor). Lysates were homogenized 20 times using a Dounce homogenizer (pestle B) (Kimble Chase) and incubated with micrococcal nuclease (Thermo Scientific, Waltham, MA, USA). Cell extracts were collected by centrifugation at 14,000 rpm at 4 °C for 20 min. Cell extracts were incubated with antibodies (anti-Flag M2 antibody, F3165, Sigma Aldrich, Saint Louis, MO, USA) overnight at 4 °C with rotation. The next day, Protein-G Dynabeads (Invitrogen) were added to the mixture and incubated for 3 h at 4 °C with rotation. After incubation, the beads were washed three times in ice-cold wash buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.05% NP-40). Proteins were eluted in acid using IgG elution buffer (Thermo Scientific) at room temperature (RT) for 10 min with gentle vortexing. The final elution was collected and neutralized with 1/10 volume of 1 M Tris-HCl, pH 9.0.