Nuclear and cytoplasmic protein extraction kit

The lysates were obtained using RIPA buffer (Sigma, USA) premixed with a protease inhibitor cocktail kit (Thermo, USA). Nuclear proteins were extracted using a nuclear and cytoplasmic protein extraction kit (Sangon, China, #C510001). Cells were lysed for 30 minutes and then centrifuged for five minutes at 12,000 ×g at 4°C. Next, the supernatant containing protein was resolved on a 10% acrylamide‐bisacrylamide gel (EpiZyme, China, #PG112), and the proteins were transferred onto a 0.45‐μm PVDF membrane (Millipore, Germany, #IPVH00010). Membranes were blocked using protein-free rapid blocking buffer (EpiZyme, #PS108) and then were incubated with primary antibodies at 4°C overnight, followed by a 1-hour incubation with horseradish-peroxidase (HRP)-conjugated secondary antibodies at room temperature. The primary antibodies used were as follows: anti-TLR4 (1:2000, ABclonal, China, #A17436), anti-NF‐κB p65 (1:3000, Cell Signaling, USA, #8242S), anti-IkBα (1:2000, Proteintech, China, #10268-1-AP), anti-β-actin (1:1000, Beyotime, China, #AF0003), and anti-GAPDH (1:2000, Proteintech, #10494-1-AP). All experiments were performed in triplicate.

Total protein samples were prepared in radioimmunoprecipitation assay (RIPA) buffer; then, the protein concentration was determined by BCA assay (Beyotime). The total protein were separated by electrophoresis through Future PAGETM 4–12% (ACE, 11 Wells), then transferred the protein to PVDF membranes (Millipore, 0.45 µm), blocked with 8% skim milk and incubated with anti-PYGO2 (1:1000, Genetex), anti-NXK2.5 (1:1000; Invitrogen), anti-GATA4 (1:1000, Proteintech), anti-cTnT (1:1000, Abcam), anti-β-catenin (1:1000, Proteintech), anti-β-Tubulin (1:5000, ABcanol), anti-ACTA2 (1:1000, Abcam), anti-GAPDH (1:5000, ABcanol), and anti-β-ACTIN antibody (1:5000 dilution, ABcanol). The signal densities of the target protein bands were quantified and normalized to β-ACTIN or GAPDH using Image J. Nucleo-cytoplasmic isolation was performed according to the instructions of the Nuclear and Cytoplasmic Protein Extraction Kit (Sangon Biotech), followed by routine protein isolation and antibody incubation. As for the analysis of nuclear localization of β-catenin, we used the formula [(β-catenin/H3)-(Protein/GAPDH) + 1] (in both the vector and PYGO2 groups) to exclude for cytopasmic contamination.

PK-15 cells grown into six well plates, were transfected with the recombinant plasmids and incubated at 37 °C for 24 h. The nuclear and cytoplasmic protein were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Sangon Biotech, Shanghai, China) following the manufacturer’s protocol. The cells were then washed gently with PBS and collected. The cell pellets were resuspended in 100 μL cytoplasmic protein extraction buffer A, incubated on ice for 3 min, and then the preparation was spun at 1500× g for 4 min. The supernatants were transferred as cytoplasmic protein, and the nuclear pellets were resuspended using 50 μL nuclear protein extraction buffer B. We then incubated the mixture on ice for 10 min, and it was swirled to resuspend the pellets. The nuclear protein extraction was centrifuged at 12,000× g for 10 min, and the supernatants were collected as the nuclear protein.

The total protein of lung tissues and cultured cells was extracted by using RIPA buffer (P0013D, Beyotime, Shanghai, China) containing 1% protease and phosphatase inhibitor cocktail. The homogenates were centrifuged at 12,000 g for 20 min and the supernatants were collected as the total protein. The cytosolic and nuclear protein fractions of mice lungs were prepared according to the instruction of a commercial Nuclear and Cytoplasmic Protein Extraction Kit (Sangon Biotech, Shanghai, China). The protein concentration of the prepared samples was determined by using BCA protein assay kits. Equal aliquots of samples were mixed with loading buffer, and heated at 100°C for 5 min.
An appropriate amount of protein samples (10–40 μg) was separated by 7.5–15% SDS/polyacrylamide gel electrophoresis and then transferred to PVDF membranes. After blocking for 1 h with 4% skim milk in TBST buffer, the membranes were incubated with primary antibodies overnight at 4°C. Then the membranes were washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Immunoreactive bands of protein were detected by an enhanced chemiluminescence kit and exposed to X-ray films. The blots were scanned using an EPSON DS-6500 scanner, and the relative optical density of the bands was quantified by using ImageJ software.

Homogenates of epithelium were lysed in RIPA solution (Yeasen) supplemented with a protease inhibitor cocktail (Sigma‐Aldrich) at 4 °C for 1 h. The protein from epithelial mitochondria was extracted using the Mitochondrial Isolation Kit (Yeasen) according to the manufacturer's instructions. Nuclear proteins from epithelial cells were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Sangon Biotech) according to the manufacturer's instructions. Samples were clarified, denatured with SDS buffer, and boiled for 10 min. Proteins were separated by SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were immunoblotted with primary antibodies: rabbit anti‐ECSIT (Novus, 1:1000), rabbit anti‐YAP (CST, 1:1000), rabbitanti‐SCA1 (SAB, 1:1000), rabbit anti‐Histone H3 (Proteintech, 1:2000), rabbit anti‐CCND2 (SAB, 1:1000), rabbit anti‐ATP5A (Bioworld, 1:1000), rabbit anti‐UQCRC2 (Bioworld, 1:1000), rabbit anti‐SDHB (Bioworld, 1:1000), rabbit anti‐MTCO1 (Abcam, 1:1000), or mouse anti‐NDUFS8 (Santa Cruz, 1:1000) and proteins detected with appropriate secondary anti‐rabbit or anti‐mouse antibody conjugated to fluorescence. Immunoreactivity was visualized using the Odyssey Imaging System (LI‐COR Biosciences).