Affinity Purification of NUPR1 Complexes

The experimental set-up was the same described previously (Lan et al, 2020 (link)). Briefly, MiaPaCa-2 cells, expressing Flag-NUPR1 or GFP-NUPR1 or their controls, were plated in 10 cm² dishes. When MiaPaCa-2 cells expressing Flag-NUPR1 or GFP-NUPR1 reached 70% confluence, they were treated for 24 h and lysed. Equal amounts of total protein were used to incubate with 30 μL of anti-Flag M2-coated beads (Millipore Sigma, F3165) or GFP-Trap Agarose (Chromotek, GTA-10). Beads were then washed 3 times, and proteins were eluted using ammonium hydrogen carbonate buffer containing 0.1 μg/μL of Flag peptide (Millipore Sigma, F3290). Eluted proteins were collected and loaded on NuPAGE 4–12% Bis-Tris acrylamide gels according to the manufacturer’s instructions (Invitrogen). Protein-containing bands were stained with Imperial Blue (Pierce), cut from the gel, and digested with high-sequencing-grade trypsin (Promega) before MS analysis. MS analysis was carried out by LC-MS/MS using an LTQ-Velos-Orbitrap or a Q Exactive Plus Hybrid Quadrupole-Orbitrap (Thermo Fisher Scientific) coupled online with a nanoLC Ultimate3000RSLC chromatography system (Dionex). Raw files generated from MS analysis were processed using Proteome Discoverer 1.4.1.14 (Thermo Fisher Scientific).