5.5. Proteomics of Supernatant (SN), Cytoplasm (CYT), and Nuclear Extract (NE)

For the proteomics sample preparation, the s-trap system (Protifi, Huntington, NY) was employed following the manufacturers protocol with slight modifications. The precipitated proteins were dissolved in lysis buffer (8 M Urea, 0.05 M triethylammonium bicarbonate (TEAB) and 5% sodium dodecyl sulfate SDS) and diluted to obtain a protein concentration of about 1 µg/µL. Twenty µg of protein was used for each digestion. First, the sample was reduced with 20 µL dithiothreitol DTT (Sigma-Aldrich) at a final concentration of 32 mM for 10 min at 95 °C. Afterwards, 5 µL iodoacetamide IAA (Sigma-Aldrich) was added to a final concentration of 54 mM and incubated for 30 min at 30 °C in the dark. After adding 4.5 µL 12% ortho-phosphoric acid (Sigma-Aldrich) and 297 µl S- Trap buffer (90% Methanol (v/v) in H₂O and 0.1 M TEAB) the sample was loaded onto the S-Trap Filter. The S-trap filters were centrifuged at 4000 xg for 1 min to pass through all the sample and trap the proteins onto the resin and afterwards washed four times with 150 µL S-Trap buffer. Twenty µg aliquots of Trypsin/Lys-C (MS grade; Promega Corporation, Madison, WI, USA) were dissolved in 400 µL 50 mM TEAB and 20 µg of this solution was added directly onto the resin of the filter (corresponding to 1 µg Trypsin/Lys-C per sample) and incubated for 1 h at 47 °C. After finishing the digestion, the peptides were eluted with 40 µL of 50 mM TEAB followed by 40 µL of 0.2% formic acid (FA) in H₂O and 35 µL of 50% (v/v) acetonitrile (ACN) with 0.2% FA in H₂O. The peptides were dried for about 2 h with vacuum centrifugation and stored at −20 °C until LC-MS/MS measurement.