HT-29/shMLKL/MLKL–3xFlag-HA cells were cultured to confluence of 80% in 60 of 15-cm culture dishes. Cells were treated with TNFα, Smac mimetic, and z-VAD for 6 hours before being collected by scraping. Cells were then centrifuged at 1000g for 10 min to remove culture medium. The collected cell pellet was washed with cold PBS three times to completely remove any residual culture medium. PBS containing 2 mM DSP cross-linker was then used to resuspend the cell pellet. The reaction was done at room temperature for 30 min before being quenched by tris-HCl buffer (pH 7.4) at a final concentration of 20 mM. Cells were then pelleted again and lysed on ice in Flag lysis buffer [150 mM NaCl, 20 mM tris-HCl (pH 7.4), 0.5% Triton X-100, protease inhibitor cocktail, and phosphatase inhibitor cocktail]. The cell suspension was centrifuged at 500g for 10 min, after which the supernatant was further centrifuged at 13,000g for 10 min. The resulting supernatant was then centrifuged again at 100,000g for 60 min. The pellet collected from this 100,000g centrifugation (P100) was then dissolved in Flag lysis buffer plus 0.5% SDS by rotating overnight to allow thorough lysis. The lysate was then centrifuged again at 100,000g for 60 min to remove any insoluble particles. The supernatant from the final centrifugation was collected, and SDS was added to a final dilution to 0.1% before the supernatant was used for Flag and HA affinity pull-down assays.

Cell lysates were incubated with 50 μl of anti-Flag agarose overnight at 4°C and washed with Flag lysis buffer three times before being eluted in Flag peptide (0.1 mg/ml) for 6 hours. The Flag elute was then incubated with 20 μl of anti-HA agarose overnight at 4°C before being washed three times again in Flag lysis buffer, followed by elution in HA peptide (0.5 mg/ml) for 6 hours. The final elute was then denatured by boiling for 10 min in 1× SDS protein loading buffer containing β-mercaptoethanol. It was then separated by SDS-PAGE followed by silver staining.

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