Mabg flapa

Bacterial extracts were separated on a 12% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gel and blotted on polyvinylidene difluoride (PVDF) membrane blocked with 5% milk in TBS with 0.1% [v/v] Tween 20. The flagellin protein was detected with a monoclonal α‐flagellin antibody specific to purified flagellin from P. aeruginosa (mabg‐flapa, Invivogen, Toulouse, France) used in a 1 : 1000 dilution and α‐mouse‐HRP (Sigma‐Aldrich) used in a 1 : 10 000 dilution as secondary antibody. Visualization was achieved with chemiluminescent ECL pico solution (Thermo Fisher Scientific) and imaged with a LAS 4000 ImageQuant system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Equal loading of protein was determined by Coomassie Brilliant Blue staining of the blotted membrane.

Flagellin purified from P. aeruginosa was purchased from InvivoGen. DOTAP liposomal transfection reagent was purchased from Sigma-Aldrich. Lipopolysaccharide (LPS), ATP, nigericin, pyocyanin, and proteinase K were purchased from Sigma-Aldrich. N-3-oxo-dodecanoyl-l-homoserine lactone (3-oxo-C12-HSL) and N-butyryl-l-homoserine lactone (C4-HSL) were obtained from Cayman. Antibodies were acquired to detect mouse caspase-1 (Adipogen, 20B-0042), mouse IL-1β (R&D, AF-401-NA), mouse IL-6 (Cell Signaling, 12912), mouse NLRP3 (Adipogen, 20B-0014), mouse ASC (Santa Cruz, SC-22514-R), and Flagellin (InvivoGen, mabg-flapa).