Sds page electrophoresis

Cells and culture supernatants, or whole colon homogenates, were incubated with cell lysis buffer (20 mM Tris HCl (pH 7.4), 200 mM NaCl, 1% Nonidet P-40), and denatured in Laemlli buffer by boiling for 10 min. Proteins were separated by SDS-PAGE electrophoresis (Thermo Scientific) after which proteins were transferred to membranes using turbo (7 min) blotting. Blocking and antibody incubation were performed in PBS supplemented with 0.05% Tween20 (vol/vol) and 3% non-fat dry milk. The membranes were incubated overnight at 4˚ C with primary antibodies against caspase-1 (1:1000; Adipogen, AG-20B-0042-C100), IL-1β (1:2000; GeneTex, GTX74034), GSDMD (1:1000, Abcam, ab209845), anti-cCasp3 Asp175 (1:1000, Cell Signaling, 9661S), caspase-3 (1:1000, Cell signaling, 9662S), cleaved-caspase-8 (Asp387) (D5B2) (1:1000, 8592S, Cell signaling), MLKL (1:1000, Sigma-Aldrich, MABC604) or pMLKL (Ser345) (D6E3G) (1:1000, Cell signaling, 37333). After washing, membranes were incubated with HRP-conjugated anti-mouse, anti-rabbit or anti-rat antibodies (1:5000; Jackson ImmunoResearch Laboratories, 115-035-146, 111-035-144 and 112-035-143) or were incubated with the directly labeled primary antibody β-actin-HRP (1:10000; Santa Cruz) for up to 3 h. Proteins of interest were detected by the enhanced SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).

Tissue samples were lysed by a radio-immunoprecipitation assay (RIPA) buffer. A bicinchoninic acid assay (Invitrogen, CA) was used to determine the protein concentration in the tumors according to the manufacturer’s protocols. ELISA assay was performed according to the manufacturer’s protocols after appropriate titration. For western blot analysis, the protein solution was diluted by sample buffer (4×) containing reducing reagent and heated at 95 °C for 5 min. Protein was separated by 4–12% SDS-PAGE electrophoresis (Invitrogen), and then transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with 3% bovine serum albumin at room temperature for 1 h and then incubated with primary antibodies overnight at 4 °C. The membranes were washed and further incubated with a secondary antibody (appropriate diluted) at room temperature for 1 h, and then detected using the Pierce ECL Western Blotting Substrate (ThermoFisher Scientific, IL). GAPDH was used as the control.

Tissue samples were lysed with a Radio‐Immunoprecipitation Assay buffer and the extracted protein were quantified by BCA assay (Invitrogen, CA) following the manufacturer's protocols. The protein solution was mixed with 4 × buffer containing reducing reagent and heated at 95 °C for 5 min. Then, protein samples were separated by 4–12% SDS‐PAGE electrophoresis (Invitrogen), after which they were transferred to polyvinylidene difluoride (PVDF) membranes (Bio‐Rad). PVDF membranes containing proteins were blocked with 3–5% bovine serum albumin at room temperature for 1 h and then incubated with primary antibodies at 4 °C overnight. GAPDH was used as a control. After being washed and incubated with secondary antibodies, PVDF membranes were detected using ECL western blotting substrate (Thermo Fisher Scientific, IL).