Mouse anti il 1β antibody

150,000 cells were seeded on cover slips and treated as indicated in figure legends. At the end of stimulation/treatment time, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 15 min and blocked in 2% BSA (Fisher). Cells were labelled with the following primary antibodies against specific targets using 1:200 dilutions: rabbit anti-IL-8 antibody (Biorad Biosciences), mouse anti-IL-1β antibody (Invitrogen), mouse anti-MCP-1 antibody (Abcam), mouse anti-NF-κB p65 antibody (Santa Cruz Biotechnology), mouse anti-GM-130 antibody (BD Biosciences) or rabbit anti-GGA2 antibody (BD Biosciences). Primary antibodies were labelled with the following fluorescently conjugated secondary antibodies diluted 1:500: Alexafluor 555 donkey anti-rabbit and Alexafluor 488 donkey anti-mouse antibodies. (Invitrogen). F-actin was labelled with phalloidin-iFluor 405 (Abcam) and nuclei with the DNA stain, DAPI (Sigma). Cover slips were mounted with Mowiol mounting media (Gift from Dr. A. Simmonds, University of Alberta) and imaged with a Zeiss LSM 700 confocal microscope (Zeiss) using a 63x objective (NA 1.4) and images were acquired using Zen software (Zeiss). Images were subsequently processed with ImageJ Software (U.S. National Institutes of Health).

BEAS-2B cells were pretreated with ML141, monensin or vehicle control (DMSO) for 1 h. Cells were subsequently stimulated with TNF-α for 4 h, fixed with 4% paraformaldehyde and resuspended in flow cytometry buffer composed of 0.1% Saponin (Sigma), 1% BSA (Fisher), PBS (Sigma). Unstained cells were treated with primary antibodies only and used for gating to eliminate non-specific signals. Cells were stained with rabbit anti-IL-8 antibody (Biorad Biosciences) and mouse anti-IL-1β antibody (Invitrogen) and Alexafluor 555 donkey anti-rabbit and Alexafluor 488 donkey anti-mouse antibodies (Invitrogen). BD LSRFortessa™ Cell Analyzer was used to detect fluorescence within cells. BD FACSDiva™ Software was used for data acquisition and analysis.