Polyacrylamide gel

Western blot analysis was carried out as previously described^{10 (link)}. The BCA Protein Assay was used to determine the protein concentration. Forty micrograms of protein were dissolved in 4 to 15% Bio-Rad polyacrylamide gels and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Germany). The membranes were blocked in Odyssey (LI-COR) blocking buffer for 1.5 hours at room temperature and then incubated overnight in the corresponding primary antibody. Then, the membranes were incubated with the corresponding secondary antibodies. Enhanced chemiluminescent immunoblotting (ECL, Bio-Rad, Japan) was used to visualize the membranes. Western blot quantification was performed using Image Lab software.

Tissue was placed in a 1.5 ml centrifuge tube and 10 times the volume of tissue lysate were added, as well as the protease and phosphatase inhibitors followed by homogenization with an ultrasonic shredder. The homogenate was allowed to stand for half an hour at room temperature and then centrifuged at 14000 rpm for 15 min to remove debris and nuclei. The supernatant was boiled for 10 min in a water bath and then put on ice for 5 min; the precipitated proteins were removed by centrifugation 14000 rpm for 5 min. The supernatant was saved for analysis. Use the Bio-Rad DC Protein Assay to determine the protein concentration. Forty micrograms of protein were resolved on 4 to 15% Bio-Rad polyacrylamide gels and then transferred onto polyvinylidenefluoride (PVDF) membranes (Millipore, Germany). The membranes were blocked in Odyssey (LI-COR) blocking buffer for 1.5 hours at room temperature and then incubated overnight in the corresponding primary antibody. After that, the membranes were incubated with corresponding secondary antibodies. Finally, use enhanced chemiluminescent immuno blotting (ECL, Bio-Rad, Japan) to make the membranes visualized. Western Blot quantification was performed using Image lab software.

Mouse and human lung tissues were homogenized in RIPA lysis buffer (Beyotime) containing a protease inhibitor cocktail (Roche), and equal amounts of lysates were separated on 10% polyacrylamide gels (Sigma‐Aldrich) and transferred onto polyvinylidene difluoride membranes as previously described.¹⁷ Target protein analysis was performed as described using appropriate primary antibodies, followed by probing with the corresponding horseradish peroxidase‐conjugated secondary antibodies. The reactive bands were visualized using ECL reagents (Servicebio), and the band intensities were analyzed using ImageJ software.

Lung tissues and cultured cells were homogenized in T-PER Tissue Protein Extraction Reagent (Thermo Scientific), and RIPA lysis buffer was added with phenylmethylsulfonyl fluoride for extraction of total proteins (Beyotime Institute of Biotechnology, Shanghai, China; PMSF, Sigma-Aldrich). A total of 80 μg of protein extracts were separated on 10% polyacrylamide gels (Sigma-Aldrich) and transferred onto polyvinylidene difluoride membranes (0.2 μm, Immobilon, ISEQ00010). Protein bands were next incubated with indicated primary antibodies for analysis of protein levels as described previously (37 (link)).
The following primary antibodies were used: antibody against fibronectin (Abcam, ab45688); antibody against collagen I (ABclonal, A1352); antibody against α-SMA (Abcam, ab32575); antibody against UHRF1 (Santa Cruz, H-8, sc-373750); antibody against beclin 1 (ABclonal, A7353); antibody against Dnmt1 (Santa Cruz, sc-271729); antibody against TEAD1 (ABclonal, A13366); antibody against TEAD4 (ABclonal, A4151); antibody against YAP (Proteintech, 13584-1-AP); and antibody against GAPDH (ABclonal, AC002).

Lung tissues of mice were homogenized in radioimmunoprecipitation assay lysis buffer (Servicebio, Wuhan, China) containing a protease inhibitor cocktail (Roche, IN, USA), and equal amounts of lysates were separated on 10% polyacrylamide gels (Sigma-Aldrich) and transferred onto polyvinylidene difluoride membranes. The membranes were next probed with the indicated primary antibodies for the analysis of protein levels as previously described (21 (link)). The reactive bands were visualized using ECL plus reagents (Servicebio, Wuhan, China), and the relative intensities of bands were analyzed using ImageJ software.

Protein molecular weights were determined qualitatively by SDS-PAGE as described previously [25 (link)]. Briefly, loading buffer was added to whole cell lysates or purified fusion proteins and boiled for 3–5 min. Then samples were separated by SDS-PAGE with a 10% (w/v) polyacrylamide gel (Sigma, St. Louis, MO, USA) and stained by Coomassie brilliant blue.