Western Blot Analysis Protocol

For western blot analysis cells were washed with phosphate-buffered saline (PBS), harvested by using a cell lifter, and lysed in Radioimmunoprecipitation assay (RIPA) buffer with complete Mini EDTA-free protease inhibitor tablets (Roche) and phosphatase inhibitor cocktail PhosSTOP (Roche). The protein concentration was quantified using the BCA Assay (ThermoFischer Scientific) as described earlier [37 (link)]. 20 μg protein lysate were separated by SDS-gel electrophoresis using a NuPAGE™ 4–12% Bis-Tris protein gel and transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (all ThermoFischer Scientific). As protein standards, 10 μl Spectra Multicolour Broad Range (ThermoFisher Scientific) and 1 μl MagicMark™ XP Western Protein Standard (ThermoFisher Scientific) were used. For detection, the membranes were incubated with WesternBright Sirius HRP substrate (Advansta). Except for the membranes displayed in S2C Fig, all signals were detected by a Microchemi chemiluminescence system (DNR Bio-Imaging Systems). S2C Fig had been digitalised by using an Odyssey CT (LI-COR). Antibodies used are listed in S2 Table. Densitometric analysis of experiments was made with the Image-Studio Lite 5.2 software (LI-COR). Uncropped western blot images are displayed in the supplementary files. Raw images files are displayed in S1 Raw images.