Reaction buffer

293FT cells (Invitrogen) were transfected with expression vectors for wild-type or mutant NHLRC2-HA using polyethyleneimine “MAX” transfection reagent (Polysciences, Warrington, PA, USA) and then lysed in Cell Lysis Buffer (BioVision, Milpitas, CA, USA). Aliquots of lysates were incubated with 1 unit of recombinant active caspases (BioVision) in 1 × Reaction Buffer (BioVision) at 37 °C for 1 h. Reactions were terminated by adding Laemmli sample buffer and boiling at 95 °C for 5 min. Cleavage was evaluated by immunoblotting using an anti-NHLRC2 antibody.

Caspase-3 activity was determined by using a caspase-3 assay kit (PharMingen, San Diego, CA) according to the manufacturer's instructions. The cells (1×105) were homogenized in a lysis buffer containing 0.1% CHAPS, 50 mM HEPES (pH 8.0), 12.5 mM NaCl, 0.1 mM EDTA, and 5 mM DTT freshly added. The whole cell lysates (40 mg) were incubated with 20 mM of colorimetric substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) in a reaction buffer (BioVision) containing 5 mM DTT at 37°C for 1 h. pNA was released from the substrate upon cleavage by DEVDase. The yellow color produced by free pNA was monitored by a spectrophotometer at 405 nm. The amount of yellow color produced upon cleavage was proportional to the amount of DEVDase activity.

Viruses, heat-inactivated at 96 °C for 10 minutes, were incubated with active human recombinant caspase-3 (with or without caspase-3 inhibitor) or active human recombinant granzyme B, in reaction buffer (BioVision) at 37 °C for 1 hour or as stated in the text. Samples were then processed for immunoblotting as described above to analyze for cleavage of N protein.