Nupage 4 12 bis tris acrylamide gels

Protein extracts from each biological replicates were loaded on NuPAGE 4–12% bis–tris acrylamide gels (Invitrogen). Running of protein was stopped as soon as proteins stacked in a single band, stained with InstantBlue (Expedeon), cut from the gel and digested with trypsin protease, MS Grade (Pierce) as described previously (Shevchenko et al, 1996). Mass spectrometry analysis was carried out by LC−MS/MS using similar settings as described above except that samples were measured in “Top Speed” data‐dependent acquisition mode. Statistical significance was analyzed from the three replicate samples by two‐tailed t‐test without adjusting for multiple hypothesis testing.

The experimental set-up was the same as described previously¹¹ . 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 (MilliporeSigma, 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 (MilliporeSigma, 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).

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).