Flag sepharose beads

Collected cells were resuspended in cold lysis buffer (50 mM TrisHCl at pH 7.5, 150 mM NaCl, 50 mM NaF, 5 mM sodium pyrophosphate, 50 mM β-glycerol-phosphate, 0.1% NP 40, and 10% glycerol) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitors mixture (Thermo Scientific, Waltham, MA, USA). Cells were lysed using glass beads, and the protein concentration of the extracts was measured at 280 nm and normalized with lysis buffer. For immunoprecipitation, 200 µL of extracts were incubated with 40 µL of FLAG-Sepharose beads (Sigma-Aldrich) overnight at 4 °C. Beads were extensively washed with alkaline phosphatase buffer (50 mM Tris-HCl at pH 7.5, 100 mM NaCl, 10 mM MgCl₂, and 1 mM DTT, adjusted to a pH of 7.9) and finally resuspended with 400 µL of the same buffer. When indicated, 15 µL of alkaline phosphatase from calf intestine (ALP) or 40 µL of 100 mM sodium orthovanadate (Na₃VO₄), as a phosphatase inhibitor, to a final concentration of 10 mM were added. Samples were incubated at 37 °C for 1 h and centrifugated at 3000 rpm for 1 min. We added 2× SDS loading buffer. Proteins were boiled for 5 min and then analyzed by SDS-PAGE and immunoblotting.

Label‐free GFP pulldowns were performed in triplicate as previously described (Kloet et al, 2018). 2 mg of nuclear extract was incubated with 7.5 μl GFP‐Trap beads (Chromotek) or 15 μl FLAG‐sepharose beads (Sigma) and 50 μg/ml ethidium bromide in Buffer C (300 mM NaCl, 20 mM HEPES/KOH, pH 7.9, 20% v/v glycerol, 2 mM MgCl₂, 0.2 mM EDTA) with 0.1% NP‐40, protease inhibitors and 0.5 mM DTT in a total volume of 400 μl. After incubation, 6 washes were performed: 2 with Buffer C and 0.5% NP‐40, 2 with PBS and 0.5% NP‐40, and 2 with PBS. Affinity purified proteins were subject to on‐bead trypsin digestion as previously described (Smits et al, 2013). In short, beads were resuspended in 50 μl elution buffer (2 M urea, 50 mM Tris, pH 7.5, 10 mM DTT) and incubated for 20 min in a thermoshaker at room temperature. After addition of 50 mM iodoacetamide (IAA), beads were incubated for 10 min in a thermoshaker at room temperature in the dark. Proteins were then on‐bead digested into tryptic peptides by addition of 0.25 μg trypsin (Promega) and subsequent incubation for 2 h in a thermoshaker at room temperature. The supernatant was transferred to new tubes and further digested overnight at room temperature with an additional 0.1 μg of trypsin. Tryptic peptides were acidified and desalted using StageTips (Smits et al, 2013) prior to mass spectrometry analyses.