Phalloidin ifluor 405

Rabbit anti-phospho-STAT5 (RRID:AB_823649), rabbit anti-CISH (RRID:AB_11178524) and rabbit polyclonal anti-TFEB (RRID:AB_11220225) were all bought from Cell Signaling Technology, Leiden, The Netherlands. Phalloidin-iFluor 647 and Phalloidin-iFluor 405 were obtained from Abcam, Cambridge, United Kingdom. Goat anti-rabbit IgG (H + L) AlexaFluor647 conjugate (RRID:AB_2536101) was purchased from ThermoFisher Scientific, Breda, The Netherlands.. Anti-human CD11b-PE (RRID:AB_395789) and anti-human CD1a-BV605 (RRID:AB_2741933) were acquired from BD BioSciences, Vianen, The Netherlands and anti-human CD14-FITC (RRID:AB_830677) and anti-human CD163-AF647 (RRID:AB_2563475) from BioLegend, San Diego, CA, USA.

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).

The salivary glands of mTomato/mGFP mice were fixed in 4% formaldehyde overnight at 4°C and then frozen in liquid nitrogen and stored at −80°C. Cryosections were cut using a Leica CM3050S cryostat, after which samples were mounted in Fluoromount G on a glass slides and covered with a #1.5 coverslip. Immunostaining was performed as follows. Samples were incubated (1) in 10% fetal bovine serum (FBS) and (2) 0.02% saponin in FBS (blocking solution) for 30–45 min at room temperature with primary goat antibodies (NKCC1, 1:00; N-16, Santa Cruz) at 4°C overnight; (3) with secondary antibodies anti- goat Alexa Fluor 488 in blocking solution at 4°C for 30 min; and (4) if needed, with phalloidin-iFluor 405 (Abcam) for 30–60 min at room temperature. Finally, samples were mounted in Fluoromount G on a glass slide and covered with a #1.5 coverslip. Confocal images were acquired using a FluoView 1000 (Olympus).