Western blot

Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Nuclear and cytoplasmic extracts of OK cells which were transiently transfected with gene expression vectors or siRNA for gene suppression were prepared for western blot analysis as follows: 20 ‐ 40 µg protein was mixed with 6 x Laemmli buffer and H₂O followed by vortexing and an incubation step of 5 min at 95°C. Samples were loaded on a 10 % polyacrylamide gel with stacking gel and electrophoresis was performed for 20 min at 100 Volt (V) followed by 60 ‐120 min at 120 V in a Mini‐PROTEAN® Tetra Cell Systems (Biorad). Precision Plus Protein™ WesternC™ Blotting Standards was used to detect protein expression according to molecular weight. Proteins were then transferred to a PVDF membrane (GE Healthcare Europe GmbH) with the standard program (30 min at 25 V) of a Trans‐Blot^® Turbo™ Transfer System (Bio‐Rad). After blotting, the membranes were blocked using Tris‐buffered saline (TBS) containing 0.1% Tween‐20 and 5 % non‐fat dry milk and membranes were further incubated with primary antibody overnight at 4°C (HNRNPK: sc‐28380, Santa Cruz; UAP56, kind gift from Niels Gehring, Institute for Genetics, University of Cologne (Gromadzka et al., 2016)). After extensive washing with TBST, membranes were incubated with the corresponding secondary HRP‐coupled antibody and protein bands were visualized by enhanced chemiluminescence (ECL) using Pierce^® ECL Western Blotting Substrate and SuperSignal™ West Femto Maximum Sensitivity Substrate (ratio 4:1) on an ECL ChemoCam Imager workstation (INTAS Science Imaging Instruments GmbH). Semi‐quantification of protein expression was performed using ImageJ software.