Western blot
Total protein was extracted from
murine lung tissues using Radio
Immunoprecipitation Assay (RIPA) lysis buffer (P0013b, Beyotime,
Shanghai, China). Protein concentrations of samples were measured using
the BCA Protein Analysis kit (23225, Thermo Fisher Scientific, Waltham,
MA, USA). Sodium dodecyl sulfate-polyacrylamide gels (8% or 10%) were
used to separate protein samples, which were then transferred to
polyvinylidene fluoride (PVDF)
membranes. Blocked for 1 h at room temperature by 5% dedicated skimmed
milk, membranes were then incubated overnight with primary antibodies at
4℃ and then incubated with secondary antibodies for 1 h at room
temperature. Protein signals were detected by a Tanon automated
chemiluminescence fluorescence image analysis system (5200, Tanon,
Shanghai, China) with β -actin as an internal reference. Relative
protein expression levels were analyzed by ImageJ software and
calculated as gray-scale values with band gray value/internal parameter
(β -actin) gray value.