Western Blot Analysis of Pneumococcal Toxin

The supernatant from S. pneumoniae strains were mixed with 2% SDS-sample buffer, heated at 99 °C for 3 min, separated by SDS-PAGE using 12.5% gels (Gellex International, Tokyo, Japan), and transferred to polyvinylidene difluoride membranes (Merck Millipore). The membranes were incubated with blocking reagent (Nacalai Tesque) to block nonspecific binding and probed with the anti-PLY antibody (Abcam) diluted in Tris-buffered saline containing 0.05% Tween 20 (TaKaRa, Shiga, Japan). The membrane was then incubated with a HRP-conjugated secondary antibody (Cell Signalling Technologies, Danvers, MA, USA) in Tris-buffered saline containing 0.05% tween 20. The membrane was treated with HRP substrates (GE Healthcare) and analysed by chemiluminescence detector (Fujifilm, Tokyo, Japan). As a loading control, whole-cell lysates of S. pneumoniae strains prepared through the process of bacterial-supernatant preparation were immunoblotted with an antibody specific for pneumococcal mid-cell-anchored protein Z, which is a key membrane protein involved in bacterial-cell division and morphogenesis55 (link).