Western Blot Analysis of Lung Tissue

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).