Cell Lysis and Western Blotting Protocol

Sample preparation and western blotting were performed as previously described [21 (link)]. Cells, including detached ones, were freshly lysed in cold caspase lysis buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂ and 1% NP-40) reconstituted with 0.25 mM AEBSF and Halt Protease and Phosphatase Inhibitor Cocktail (78440, Thermo Scientific). Cell debris was removed by high-speed centrifugation for 20 min at and 4 °C, and protein concentration was quantified with Pierce BCA Protein Assay Kit (23225, Thermo Scientific). Briefly, 25 μg of protein were resolved on Bolt 4–12% Bis-Tris Plus Gels (NW04125BOX, Invitrogen) under reducing conditions according to manufacturer’s instructions. After transfer onto PVDF membranes (IPVH00010, Millipore) using Criterion blotter (1704070, Bio-Rad), blocked membranes were incubated overnight at 4 °C with the indicated primary antibodies, washed and stained with secondary antibodies for 1 h at RT. Images were detected with WesternBright Quantum HRP substrate (K-12042-D10, Advansta) on LAS-3000 Imager (Fujifilm) and processed using ImageJ software with brightness/contrast adjustment applied to an entirely digital image if necessary.