2.6. Immunofluorescence Analysis of HMGB1 Translocation

Cells were seeded in a 4-well Chamber Slide (Thermo Scientific), treated with 25 nM rapamycin or DMSO for 12 h, or stimulated with HBSS alone (Thermo Scientific) for 3 h, and each group was recovered in growth media with reduced FBS (10%) for 12 h. And then, the cells were washed twice in PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. The fixed cells were treated with 0.1% of Triton X-100 for 15 min, then washed with PBS for another three times. After blocking with 2% bovine serum albumin (BSA) for 1 hr at room temperature, the cells were incubated with rabbit anti-rat HMGB1 antibody (Abcam, 1 : 250) overnight at 4°C. After washing the cells with PBS, Cy3-conjugated goat anti-rabbit secondary antibody (1 : 500, Invitrogen Molecular Probes) was applied for 1 hr at room temperature. The cells were also counterstained with {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342 staining (Sigma). The stained cells were imaged using Nikon A1 confocal microscopy (Nikon).