Lf pa0018

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).

Western blot analysis was performed as using changes in protein expression. Briefly, proteins extracted from the hairless mouse skin were separated for 3 h using 15% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA) for 1 h at 90 V. The membranes were then incubated overnight at 4° C in 5% skim milk containing anti-GAPDH (LF-PA0018; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-TLR2 (AbFrontier, AB24192), mouse anti-NF-κB, (Santa Cruz Biotechnology, SC-71675), mouse anti-TNF-α (AbFrontier, AB1793), mouse anti-IL-1β (AbFrontier, AB1413), and mouse anti-cathelicidin (AbFrontier, AB93357). The membranes were then washed in Tris-buffered saline containing Tween 20 and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies LF-SA5002 goat anti-rabbit IgG and LF-SA5001 goat anti-mouse IgG. The blots were developed using a Western Blot Detection kit (AbFrontier, LF-QC0103) [45 (link)].