Detection of apoptosis by Western blot analysis

SW10 mouse Schwann cells, L929 mouse fibroblasts, J774A.1 mouse macrophage, C2C12 mouse myoblast, Neuro-2a mouse neuroblast, sNF96.2 human Schwann cells and HUVEC human endothelial cells were cultured for 24 hrs. Synthetic mycolactone A/B with a final concentration of 300 ng/ml, 30 ng/ml, or 3 ng/ml was then added and the cells were incubated further. Floating and adhered cells were collected at 12, 24 and 48 hrs time points. Ethanol similarly diluted with culture media to a final ethanol concentration of 300 ng/ml was used as the negative control. In addition, actinomycin-D (Sigma) diluted with culture medium for 24 hrs was used as the positive control. Following the bicinchoninic acid assay (BCA assay), Western blot analysis was performed using rabbit anti-cleaved caspase-3 (Cell Signaling #9661), rabbit anti-caspase-3 antibody (Cell Signaling #9662), mouse monoclonal anti-histone H2A.XS139ph (phospho Ser139) antibody (GENETEX, Inc. GT2311), and mouse monoclonal anti-α-tubulin (Sigma T-9026) as an internal control. Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (7076) and goat anti-rabbit IgG (7074), purchased from Cell Signaling, were used as secondary antibodies. Immunoreactive bands were visualized using a chemiluminescence reagent Immuno Star LD (Wako).