Western Blot Analysis Protocol

Cell harvest, cell lysis, protein determination, and western blot were performed as previously described [18 (link)]. 20 µg protein lysate electrophoresis were separated by SDS-gel-electrophorese using NuPAGE™ 4–12% Bis-Tris protein gels and subsequently transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (all Thermo Fisher Scientific, Waltham, MA, USA). 5 µL Spectra Multicolour Broad Range (Thermo Fisher Scientific, Waltham, MA, USA) protein standard and 1 µL MagicMark™ XP Western Protein Standard (Thermo Fisher Scientific, Waltham, MA, USA) were used. For detection, the membranes were incubated with WesternBright Sirius HRP substrate (Advansta, San Jose, CA, USA), and all signals were detected by a Microchemi chemiluminescence system (DNR Bio-Imaging Systems, Jerusalem, Israel). Densitometric analysis of experiments was performed with the Image-Studio Lite 5.2 software (LI-COR, Lincoln, Dearborn, MI, USA). Used antibodies are listed in Table 2. Uncropped western blot images are displayed in the Supplementary Files, Figures S2–S10.