Immunofluorescent assays

Subcellular localization and translocalization of NFAT5 were assessed by anti-NFAT5 antibody (Cat: ab3446; Abcam). DCs were either infected by E. hellem (30:1/spores: cells) or S. aureus (5:1/DCs: S. aureus), or stimulated by NaCl (110 mM). Cells were fixed by paraformaldehyde and permeabilized with Triton X-100. After washing and blocking, samples were incubated with NFAT5 antibody, followed by Alexa Fluor 594–conjugated secondary antibody (Thermo Fisher Scientific). Samples were then washed in PBST and stained for 5 min in DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich) for nucleus labeling. Samples were imaged using an Olympus FV1200 laser scanning confocal microscope (Olympus).