Mini protease inhibitor mixture

Cells were lysed in 25 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, and 0.1% NP‐40 buffer containing Mini Protease Inhibitor Mixture (Roche) and sodium fluoride (Sigma) by a quick freezing and thawing step. Flag‐Pyrin was immuno‐precipitated using anti‐Flag M2 affinity gel (Sigma). Proteins were separated by SDS–PAGE on precast 4–15% acrylamide gels (Bio‐Rad) and transferred to TransBlot® Turbo™ Midi‐size PVDF membranes (Bio‐Rad). Antibodies used were mouse monoclonal anti‐FLAG^® (Sigma‐Aldrich, clone M2; 1:1,000 dilution), anti‐Pyrin (Adipogen, AL196, 1: 1,000 dilution), anti‐phospho S242 Pyrin (Abcam, ab200420; 1:1,000 dilution; Gao et al, 2016), and anti‐PKN1 (Becton Dickinson, BD 610687; 1:1,000 dilution). Cell lysates were reprobed with a mouse monoclonal antibody anti‐β‐actin (clone C4, Millipore; 1:5,000 dilution). Full Western blot images are presented in Source Data.

Cells were lysed in 25mM Tris HCl, 150mM NaCl, 1mM EDTA and 0.1% NP-40 buffer containing Mini Protease Inhibitor Mixture (Roche) and sodium fluoride (Sigma) by a quick freezing and thawing step. Flag-Pyrin was immuno-precipitated using anti Flag M2 affinity gel (Sigma). ASC was cross-linked in the insoluble pellet using DSS (Disuccinimidyl suberate, ThermoFisher #21655) 2 mM (1 h at 37°C). Proteins were separated by SDS/PAGE on precast 4–15% acrylamide gels (Bio-rad) and transferred to TransBlot® Turbo^™ Midi-size PVDF membranes (Bio-rad). Antibodies used were mouse monoclonal anti-FLAG® (Sigma-Aldrich, clone M2; 1:1,000 dilution), anti-Pyrin (Adipogen, AL196, 1: 1,000 dilution), anti-phospho S242 Pyrin (Abcam, ab200420; 1:1,000 dilution) (Gao et al., 2016 (link)), anti-human Caspase-1 (Santa Cruz, sc515, 1: 1,000 dilution), anti-human GSGMD (sigma, HPA044487, 1: 1,000 dilution), anti-human IL-1β (Cell signaling, #12703, 1: 1,000 dilution), anti-ASC (Santa Cruz, sc22514R, 1:1,000 dilution). Cell lysates were reprobed with a mouse monoclonal antibody anti-β-actin (clone C4, Millipore; 1:5,000 dilution).