Quantitative Western Blot Analysis Protocol

As described earlier, subcellular fractionation, cell harvest, protein determination, and western blot were performed [30 (link)]. SDS-gel separated 20 µg protein lysate electrophorese using NuPAGE™ 4–12% Bis-Tris protein gels and transferred to a nitrocellulose membrane using the iBlot dry blotting system (all Thermo Fisher Scientifc, Waltham, MA, USA). Additionally, 5 µL Spectra Multicolour Broad Range (Thermo Fisher Scientifc, Waltham, MA, USA) protein standard and 1 µL MagicMark™ XP western protein standard (Thermo Fisher Scientifc, Waltham, MA, USA) were used. For detection, the membranes were incubated with WesternBright Sirius HRP substrate (Advansta), and signals were detected by a Microchemi chemiluminescence system (DNR Bio-Imaging Systems, Ha-Satat, Israel). The antibodies used are listed in Table 2. Densitometric analysis of experiments was performed with the Image-Studio Lite 5.2 software (LI-COR, Lincoln, NE, USA). Uncropped western blot images are displayed in the Supplementary Materials. Raw images files are displayed in S1_raw_images.