Immunoprecipitation of RIP1‐RIP3 complex and the in vitro kinase assay

Scraped jejunal mucosa was homogenized in ice‐cold complete RIPA buffer [1% Nonidet P‐40, 0.25% sodium deoxycholate, 10 mm NaF, 5 mM Na₃VO₅, 1 mm phenylmethanesulphonyl fluoride and one tablet of Complete‐Mini protease inhibitors cocktail (Roche, Penzberg, Germany) added to 7 ml of buffer immediately before use]. The lysates were centrifuged at 14,000 g for 10 min and the protein concentration of the supernatant was adjusted to 5 mg ml^–1. After pre‐cleaning with recombinant protein G agarose beads (Invitrogen), the RIP1 protein was immunoprecipitated with anti‐human RIP1 (BD Bioscience, Franklin Lakes, NJ, USA) overnight at 4°C. The protein‐antibody complexes were then incubated with protein G agarose beads for 1 h at 4°C followed by centrifugation at 14,000 g at 4°C and washing with lysis buffer twice. The pellet was dissolved in 2 × electrophoresis sample buffer containing 2% (w/v) SDS, 100 mm dithithreitol and 62.5 mm Tris/HCl (pH 6.8) at a ratio of 1:1, and subjected to 95°C in a heat block for 5 min for denaturation. Samples were used for immunoblotting of RIP3.

The immune complexes were subjected to reducing SDS‐PAGE (4‐10% polyacrylamide). The resolved proteins were then electroblotted onto Hybond‐P polyvinylidene fluoride membranes (Amersham Biosciences, Piscataway, NJ, USA). After blocking for 1 h at room temperature in 5% non‐fat dry milk, the membranes were incubated with anti‐RIP1 (1:1000; BD Bioscience) or polyclonal rabbit anti‐RIP3 (1:1000; Abcam, Cambridge, UK) as the primary antibody overnight. Membranes were washed twice with Tris‐buffered saline with 0.1% Tween 20 for 5 min, and incubated with secondary goat anti‐rabbit or anti‐mouse IgG conjugated with horseradish peroxidase (1:1000; Cell Signaling, Denver, MA, USA) at room temperature for 1 h. The antigens were revealed and the band density was quantified by photoimaging analysis (Huang et al. 2013).

Scraped mucosa to be processed for in vitro kinase assays were lysed with kinase lysis buffer (20 mm Hepes, 150 mm NaCl, 1% Triton X‐100, 5 mm EDTA, 5 mm NaF, 0.2 mm Na₃VO₄, 1 mM phenylmethanesulphonyl fluoride and Complete‐Mini protease inhibitors cocktail) followed by immunoprecipitation by anti‐RIP1‐conjugated beads as described above. The bead pellets were then incubated in kinase reaction buffer (20 mm Hepes, 5 mm MgCl₂ and 5 mm MnCl₂) supplemented with 10 μm cold ATP and 1 μCi γ‐³²P‐ATP for 30 min at 30°C. The reaction was terminated by adding the same volume of 2 × SDS sample buffer and heat denaturation at 95°C for 5 min, followed by vortex and centrifugation. Samples were resolved on 4–8% SDS‐PAGE and exposed to autoradiographic films as described previously (Huang et al. 2013).