Fusion fx5 imaging system

Protein extraction from cells, mice and humans was performed as before16 (link). Ten micrograms of protein from each sample were separated by SDS-PAGE with 10% Tris-Glycine eXtended (TGX) Stain-Free™ polyacrylamide gels (Bio-Rad, Hercules, CA, USA). Stain-Free™ gels were activated by UV transillumination for 5 min using the Fusion FX5 imaging system (Vilbert Lourmat, France). Proteins were transferred to nitrocellulose membranes (Bio-Rad) and total proteins were visualized under UV using the Fusion FX5 imaging system. Bound antibody was revealed by enhanced chemiluminescence detection using a secondary antibody coupled to ImmobilonTM Western Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA, USA). The Fusion FX5 imaging system was used for immunoblot visualization. Band intensities were quantified using ImageJ 1.6 (http://imagej.nih.gov/ij/) software and normalized to the total amount of protein per lane.

Cells were collected and lysed. Total protein was extracted and quantified using the BCA Protein Assay Kit (Solarbio), following the manufacturer’s instructions. Primary antibodies (all Abcam) used included anti-NLRP3 (1:1000), anti-cleaved caspase-1 (1:1000), anti-IL-1β (1:1000), anti-E-cadherin (1:1000), anti-vimentin (1:1000), and anti-β-actin (1:5000) and were incubated at 4 °C overnight. After washing with tris-buffered saline with Tween (TBS-T) three times, the secondary antibody (Solarbio) was added for 1 h at room temperature. The ECL substrate (Solarbio) was used to detect the expression of target proteins in the FUSION FX5 imaging system (Bio-Rad, Hercules, CA, USA).