Luminata crescendo western hrp substrate reagent

Protein extracts from cells and spleen tissues were prepared using a modified Laemmli buffer (5% sodium dodecyl sulphate, 10% glycerol, 32.9 mM Tris-HCl pH 6.8) supplemented with protease and phosphatase inhibitors (Roche). Then, 30 μg of proteins from each lysate were subjected to migration on 8–12% acrylamide gels and transferred on to polyvinylidene difluoride membranes (GE Healthcare Europe GmbH) in borate buffer (50 mM Tris-HCl and 50 mM borate) for 1 h and 45 min at constant voltage (48 V). The membranes were incubated with primary antibodies overnight at 4 °C, then washed in Tris-buffered saline–Tween 0.1% and incubated for 1 h with horseradish peroxidase (HRP)-coupled secondary antibody or beta-actin peroxidase antibody (Sigma-Aldrich). The signal was revealed using a chemiluminescent reagent (Luminata Crescendo Western HRP substrate reagent, Merck Millipore) and was detected using a ChemiDoc XRS System (Bio-Rad). Uncropped blots are presented in Supplementary Fig. 7, 8.