The lung tissue of mice was collected and digested by pre-cooled tissue protein RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein content in the sample was determined by a BCA kit after being denatured in boiling water. Proteins (~30 µg/lane) were separated by SDS-polyacrylamide gel electrophoresis on 10% gels, then transferred to PVDF membranes and blocked in 5% skim milk/TBS-0.1% Tween (TBST) solution for 1 h at room temperature. The membranes were then incubated with the following primary antibodies: Anti-TLR4 (1:1,000; cat. no. ab13556; Abcam), anti-MyD88 (1:1,000; cat. no. ab219413; Abcam), anti-NF-κB (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.) and anti-GAPDH (1:1,000; cat. no. ab9485; Abcam) overnight at 4°C. After washing with TBST three times, HRP-labeled secondary antibody (1:1,000; cat. no. ab7090; Abcam) was added to the membranes for 1 h at room temperature, then washed with PBS. ECL (GE Healthcare) was used to visualize the blots and images were captured using an ImageQuant gel imaging system (GE Healthcare Bio-Sciences). The optical density ratio of the target band was then calculated by ImageJ software (version 6.0; National Institutes of Health). GAPDH was used as the loading control.

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