Protein Fractionation and Western Blot

Protein extractions were performed as previously described (Wohlhieter et al., 2020 (link)). Extraction of separate cytoplasmic and nuclear protein fractions were performed using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher) according to the manufacturer’s protocol. Briefly, 30–50 μg of protein were mixed with NuPAGE LDS sample buffer (Invitrogen), loaded in a Bis-Tris Gel (NuPAGE, Invitrogen) and resolved. Electrophoresed protein samples were transferred with Trans-Blot Turbo RTA Mini LF PVDF Transfer Kit (Bio-Rad, Alfred Nobel Drive Hercules, CA) for chemiluminescent detection. After incubating with Pierce Starting Block (PBS) Blocking Buffer (Thermo Fisher) at room temperature for 30 min, membranes were incubated in the primary antibodies (1:1000) overnight (the antibodies’ information are in Key resources table). Secondary anti-rabbit, horseradish peroxidase-linked antibodies were purchased from CST (#7074) and detected using iBright Western Blot Imaging Systems (Thermo Fisher).