L mmli buffer

Cells for Western blot analysis were lysed in Lämmli Buffer (Bio-Rad, Hercules, CA, USA) with protease inhibitors (Thermo Fisher, Waltham, MA, USA) and sodium orthovanadate (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. Thirty micrograms of total protein were separated on ExcelGels (GE Healthcare) and transferred onto nitrocellulose membranes (Bio-Rad). After blocking, membranes were incubated with primary antibodies [cleaved caspase-3 antibody (0.5 µg/ml, #MAB835; R&D Systems, Minneapolis, MN, USA), phospho-RIPKs 1 (1:100, #65746; Cell Signalling Technology, Cambridge, UK), phospho-RIPK3 (1:200, #ab209384; Abcam, Cambridge, UK), phospho-MLKL (1:500, #91689; Cell Signalling Technology, Cambridge, UK), or glyceraldehyde 3-phosphate dehydrogenase (1:2000, #2118; Cell Signalling Technology, TNF (1 µg/ml, R&D Systems)] overnight at 4 °C. After further incubation with horseradish-conjugated goat-anti-rabbit antibody (1:10,000, #170-6515; Bio-Rad, Hercules, CA, USA), secondary antibodies were visualized with Supersignal West Dura (Thermo Fisher, Waltham, MA, USA) and signals were detected using ChemiDoc System (Bio-Rad). For blocking of the TNF antibody, 1 µg TNF antibody was pre-incubated with 10 µg recombinant TNF (R&D Systems) for 4 h at 4 °C:

Cell lysates were boiled in Lämmli buffer (Bio-Rad Laboratories Inc., Hercules, CA, USA) for 10 min at 95 °C, separated on 10% SDS-PAGE gels and transferred onto poly-vinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories Inc.). Membranes were blocked using 5% (w/v) non-fatty milk powder in TBS-T [0.1% (v/v) tween20] for 1 h and incubated with primary antibodies EPHA2 (1C11A12) or α-tubulin (TU-01) (Invitrogen, Carlsbad, CA, USA) overnight at 4 °C. Secondary antibodies (Acris Antibodies GmbH, Hiddenhausen, Germany) were incubated for 1 h, membranes were washed and submerged in chemoluminescent reagent (Millipore, Burlington, MA, USA). Finally, proteins were visualised and quantified using a LI-COR Odyssey FC Scanner in combination with Image Studio (v.5.2, LI-COR Biosciences, Lincoln, NE, USA).