Nitro blue tetrazolium substrate

As our previous studies (Yu et al., 2015 (link); Wang et al., 2017 (link)), virus-infected and mock-infected cells and/or together with drug treatment were collected at indicated times. Cells were lysed directly in sodium dodecyl sulfate (SDS) sample buffer [60 mM Tris–HCl (pH 6.8), 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue], followed by boiling for 10 min. Whole-cell lysates were further subjected to SDS–PAGE. Proteins were transferred to nitrocellulose membranes (Bio-Rad) and detected with corresponding primary and alkaline phosphatase-conjugated secondary antibody. The membranes were then reacted with 5-bromo-4-chloro-39-indolylphosphate (BCIP) and nitro-blue tetrazolium (NBT) substrate (Sigma, St. Louis, MO, United States). The following antibodies were used: anti-RIPK3 (17563-1-AP, Proteintech), anti-p-MLKL (#91689, Cell Signal), anti-MLKL (#14993, Cell Signal), anti-VP1 (GTX132346, Genetex), anti-histone and anti-HA (GenScript), and anti-tubulin (11224-1-AP, Proteintech). Anti-mouse or rabbit secondary antibodies from goat were obtained from Jackson ImmunoResearch.

Samples of virus-infected RD cells or cultural supernatant were treated with 1×loading buffer (0.08 M Tris [pH 6.8] with 2.0% SDS, 10% glycerol, 0.1 M dithiothreitol, and 0.2% bromophenol blue) and boiled at 100 °C for 10 min. After centrifugation, samples were further subjected to SDS-PAGE. Then, proteins in the SDS-PAGE gel was transferred to polyvinylidene fluoride membrane (catalog no. BSP0161; Pall) and detected with corresponding primary and alkaline phosphatase-conjugated secondary antibody. The membranes were then reacted with 5-bromo-4-chloro-39-indolylphosphate (BCIP) and nitro-blue tetrazolium (NBT) substrate (Sigma-Aldrich, St. Louis, MO, USA). Polyclonal antibody against EV71 was obtained from rabbits immunized with EV71 CC077. Anti-tubulin monoclonal antibody was purchased from Abcam (MAb; ab11323, Cambridge, UK). Mouse and rabbit secondary antibodies were obtained from Proteintech.