Anti aqp5

The lung paraffin sections were dewaxed, hydrated, antigen-repaired, and circled. The paraffin-embedded sections or primary cell slides were sealed with 10% goat serum at room temperature (22–24 °C) for 1 h, then away from light at 4 °C overnight with antibodies:anti-sox9 (1:100, Santa Cruz), anti-β-catenin (1:200, Proteintech), anti-SPC (1:100, Proteintech), and anti-AQP5 (1:100, Proteintech). Next, the goat anti-rabbit or goat anti-mouse secondary antibody (1:200, Proteintech) was used to incubate for 2 h. After DAPI was used to stain the cell nucleus, a fluorescence microscope (E800, Nikon, Japan) was used to observe protein expression.

Total proteins were separated by electrophoresis using 12% SDS-polyacrylamide Tris-Gly Novex precast gels (Thermo-Fisher Scientific, Waltham, MA, USA) and then electrotransferred to a polyvinylidene difluoride (PVDF) membrane. PVDF membrane was incubated with 5% nonfat milk in PBS-0.1% Tween 20 (PBST) for 1 h at room temperature, and then with the primary antibody anti-AQP5 (1:1000; Proteintech, Rosemont, IL, USA) and anti-Actin (1:1000; Millipore, Burlington, MA, USA) in PBST overnight at 4 °C, and finally washed in PBST for 15 min. The PVDF membrane was then incubated with anti-mouse or anti-rabbit antibody (1:3000; Cell Signaling, Danvers, MA, USA) for 1 h at room temperature and washed in PBST. PVDF membranes were incubated with Western Lighting Plus-ECL reagents (Perkin-Elmer, Waltham, MA, USA) and developed using Amersham Imager 600 (GE Healthcare, Chicago, IL, USA).

Total proteins were extracted from lung tissues using a radioimmunoprecipitation assay buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors (Biosharp, China). Their levels were quantified using a bicinchoninic acid kit. Proteins were separated with sodium dodecyl sulfate–polyacrylamide gel and transferred to polyvinylidene fluoride membranes. They were blocked in 5% skim milk at 37 °C for 1 hour and incubated overnight at 4°C with primary antibodies: anti-PTEN (1:2000; Abcam, USA), anti-AKT (1:4000; Proteintech, China), anti-p-AKT (1:4000; Proteintech, China), anti-Bcl-2 (1:1000; Proteintech, China), anti-Bax (1:2000; Abcam, USA), anti-Cleaved Caspase 3 (1:2000; CST, USA), anti-SPC (1:1000; Proteintech, China), anti-AQP5 (1:1000; Proteintech, China), anti-Claudin 4 (1:4000; Proteintech, China) and anti-Keratin 8 (1:4000; Proteintech, China). β-actin (1:10000; Proteintech, China) was used as a reference protein. The membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:10000; Proteintech, China) at 37 °C for 1 hour. Protein bands were detected with enhanced chemiluminescence detection reagents (Millipore, USA) and analyzed using ImageJ software (version 1.51; National Institutes of Health, USA).