Af5169

Sections were incubated with primary antibodies against AQP-5 (Affinity, AF5169, 1:200), SP-C (Affinity, DF6647, 1:200), LC3 (CST, 12741, 1:200) or cleaved caspase-3 (CST, 9661, 1:200) for 60 min, and therewith incubated with secondary coralite488-conjugated anti-rabbit IgG (Proteintech, SA00013-2, 1:10000) or coralite594-conjugated anti-mouse IgG antibodies (Proteintech, SA00013-3, 1:10000) for 60 min. The cells were counterstained with DAPI (Servicrbio, G1012, 1 μg/mL), and observed under a fluorescence microscope (Leica Microsystems).

The protein was extracted from liver tissues, and protein concentration was determined using the bicinchoninic acid assay. Briefly, 20 μg protein in each group was processed in 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After transferring onto polyvinylidene fluoride (PVDF) membrane, non-specific staining was blocked by 5% skim milk. The membrane was incubated with the following antibodies overnight at 4°C: rabbit anti-PBEF (1:2000, 11776-1-AP, Proteintech); rabbit anti-ERK1/2 (1:1000, 16443-1-AP, Proteintech); rabbit anti-p-ERK1/2 (1:500, bs-3016R, Bioss); rabbit anti-p38MAPK (1:1000, bs-0637R, Bioss); rabbit anti-p-p38MAPK (1:1000, bs-5477R, Bioss); rabbit anti-AKT (1:500, ab8805, Abcam); rabbit anti-p-AKT (1:1000, bs-2720R, Bioss); rabbit anti-AQP1 (1:1000, bs-1506R, Bioss); rabbit anti-AQP5 (1:1000, AF5169, Affinity); rabbit anti-ENaC (1:1000, bs-2957R, Bioss); rabbit anti-surfactant protein D (SP) (1:1000, ab220422, Abcam); and mouse monoclonal anti-GAPDH (1:2000, TA-08, ZSBio).