Ab1793

For western blot analysis, proteins collected from whole lung tissue were separated by SDS-PAGE in a 15% polyacrylamide gel for 3 h. The separated proteins were then transferred to PVDF membranes (Bio-Rad, USA) for 1 h at 90 volts. After blocking the membranes overnight at 4 °C with 5% skim milk, they were probed using anti-GAPDH (Santa Cruz Biotechnology, LF-PA0018), polyclonal mouse anti-NF-κB (Santa Cruz Biotechnology, SC-71675), monoclonal mouse anti-TNF-α (ABfrontier, AB1793), polyclonal mouse anti-IL-1β (ABfrontier, AB1413), polyclonal mouse anti-Cathelicidin (ABfrontier, AB93357) and polyclonal mouse anti-PGC-1 (Millipore AB3242). The membranes were then washed with TBST buffer and incubated with secondary antibodies (goat anti-mouse IgG (HRP) LF-SA5001-conjugated). Finally, the blots were developed using a western blot detection kit (LF-QC0103, Abfrontier)74 (link).

Lungs and visceral fat were extracted from mice, washed once with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde for 24 h at 4 °C. The fixed tissue was then dehydrated through a 50–100% ethanol series (2 h at each step) and 3 incubations (1 h each) in xylene, embedded in paraffin and cut into 4-μm-thick sections using a microtome. Sections were incubated for 30 min at room temperature with primary antibody in 5% bovine serum albumin (BSA). The primary antibodies used were polyclonal mouse anti-cathelicidin (ABfrontier, AB93357), monoclonal mouse anti-TNF-α (ABfrontier, AB1793) and polyclonal mouse anti-IL-1β (ABfrontier, AB1413). The samples were washed with tris-buffered saline containing tween (TBST buffer), incubated with secondary antibody (goat anti-mouse IgG (HRP) LF-SA5001-conjugated), and stained with hematoxylin and eosin (H&E). Stained sections were examined under a fluorescence microscope (Ix71, Olympus, Tokyo, Japan)64 (link).

RAW 264.7 cells (1 × 10⁶ cells/mL) were cultured for 12 h as described above and incubated for 6 h in the presence of S. aureus LTA (1 μg/mL) with or without CSP-4 (100 μg/mL). The cells were then washed in PBS, fixed in 4% paraformaldehyde (15 min), permeabilized using 0.5% Triton-X 100 diluted in PBS, washed again 3 times by gently shaking in PBS for 5 min each, blocked in a blocking buffer (5% bovine serum albumin in PBS), and then added to an 8-well plate (200 μL/well), in which they were incubated for 1 h with gentle shaking. Primary antibodies (TNF-α, AbFrontier, Seoul, Korea, AB1793; and IL-1β AbFrontier, AB1413; 1:50 dilution) were then added to the cells for 24 h at 4° C, after which the cells were washed 3 times in PBS (5 min/wash). A secondary antibody was applied (goat anti-mouse IgG-HRP, LF-SA 5001-conjugated; 1:200 dilution) for 1 h at 37° C, and nuclei were stained using DAPI (0.25 µg/mL). The staining was visualized under a confocal laser-scanning microscope (LSM510; Carl Zeiss; Gottingen, Germany) [45 (link)].