Ba2913

The H9C2 cells were lysed in RIPA buffer (P0013C, Beyondtime) containing 1 mM PMSF (ST505, Beyondtime). Total protein of 30 μg was subjected to electrophorese on 12–6% SDS-Page gels and transferred to PVDF membranes. Antibodies against β1AR (1:1000; cat. bs-0498R, Bioss), VEGF (1:800; cat. AF5131, Affinity), PGC-1α (1:1000, cat. bs-1832R, Bioss), and caspase3 (1:1000; cat. bs-0081R, Bioss) were used as primary antibodies. Rabbit IgG antibodies coupled to horseradish peroxidase (HRP) were used as secondary antibodies. GAPDH (1:1000; cat. BA2913, Boster) was used as loading control. An enhanced chemiluminescence (ECL) system was used for detection of protein bands.

Based on a previously reported protocol [1 (link)], the dorsal neurovascular bundle and buck fascia were removed from the TA and urethra. The tissues and fibroblasts in each group were lysed in RIPA buffer (R0010, Solarbio, China). Twelve per cent SDS-PAGE was used to separate proteins (30 μg), and the separated proteins were transferred to PVDF membranes (EMD Millipore). Appropriate primary antibodies diluted with 5% BSA were incubated with the membranes overnight at 4 °C. The primary antibodies consisted of collagen 3 (1:800, AF5457, Affinity, China), Smad7 (1: 800, AF5147, Affinity, China), elastase-2B (1: 800, orb471854, Biorbyt, China), osteopontin (1: 800, AF0227, Affinity, China) and GAPDH (1:1000, BA2913, Boster, China). After 3 washes with TBS-0.01% Tween 20, the HRP-Affinipure goat anti-rabbit IgG (H + L) (1:500, BM3894, Boster, China) was incultured with the membranes for 2 h at 25 °C. After washing, the signals were visualized using enhanced chemiluminescence reagent (D085075, Bio-Rad, China).

After isolation from tissue or cell samples, total proteins were separated via SDS-PAGE and transferred onto PVDF membranes that were blocked with an appropriate solution for 1 h at room temperature. Blots were then probed for 2 h at room temperature with primary antibodies specific for Bax (1:1000, BA0315-2, Boster), Bcl-2 (1:1200, A00040-1, Boster), Cytochrome C (1:5000, ab133504, Abcam), DNAJB4 (0.1 µg/ml, ab254641, Abcam), and GAPDH (1:10,000, BA2913, Boster). Blots were then probed for 1 h at room temperature using Goat Anti-Rabbit IgG H&L (HRP) (1: 2000, ab6721, Abcam), after which they were washed, incubated in developer solution, and exposed to X-ray film to detect protein bands.

Immunohistochemistry was performed after dewaxing and rehydration. Lung slices were pretreated with an EDTA-antigen retrieval buffer in a microwave oven, and endogenous peroxidase in tissue was quenched with 3% H₂O₂ for 10 min. Lung slices were blocked with 20% normal fetal bovine serum for 30 min at 37 °C, then incubated with primary and secondary antibodies. The primary antibodies were utilized: anti-FSTL1 (1:200, ab71548, Abcam), anti-collagen I (1:200, BA2023, BOSTER), anti-SMA (1:400, BA0002, BOSTER), anti-P62 (1:200, ab56416, Abcam), anti-LC3B (1:200, ab48394, Abcam), and anti-GAPDH (1:400, BA2913, BOSTER). The staining was performed using the PV-9000 kit (Zhongshan Golden Bridge Biotechnology Co, China). After dehydrating and mounting, slices were observed under a microscope. For the expression of each protein, three slices of IHC were used to calculate the average optical density (AOD) per sample. Quantification of IHC was also performed using ImageJ.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was selected as the loading control. Tissue was homogenized in protein extraction reagent (Keygen, China). The obtained homogenate was centrifuged at 12,000 rpm for 30 min at 4 °C. The protein samples collected from the supernatant were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) at 8% concentration and transferred to 0.45-μm polyvinylidene difluoride membranes, which were subsequently blocked with TBST buffer for 2 h at 37 °C. The membranes were probed with primary rabbit anti-TSP1 (1:1000, MA5-13,398, Thermo Fisher Scientific, USA), rabbit anti-TSP2 (1:600, PA5-97,117, Thermo Fisher Scientific, USA), rabbit anti-TSP4 (1:1000, ab156258, Abcam, USA), and rabbit anti-GAPDH (1:2000, BA2913, Boster, USA) antibodies for 12 h at 4 °C. Next, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit antibody (1:10,000, BA1054, Boster, USA) for 1 h at 37 °C. The immunoreactive bands were visualized with a chemiluminescent substrate (Thermo Fisher Scientific, USA). The optical densities (ODs) of the bands on the Western blots were quantified using Fiji software (USA). The relative expression levels of specific proteins were normalized and calculated as the optical density of the specific protein band/OD of the GAPDH band.

Lung tissues samples, washed with 0.01 M PBS, were lysed in ice-cold RIPA buffer containing sodium phosphate (20 mM, pH7.4), NaCl (150 mM), 1% NP-40, 0.1% SDS, and 0.5% deoxycholic acid with sodium orthovanadate (1 mM) and protease inhibitor cocktail (Roche). After centrifuging the samples at 12,000 g for 15 min, the supernatants were collected. The total amount of protein was quantified using the Pierce BCA Protein Assay Kit. 30 μg of total protein each were separated by 10% SDS polyacrylamide gel electrophoresis and blotted into PVDF membranes. Then the membranes were probed with primary and secondary antibodies sequentially. The primary antibodies were utilized: anti-FSTL1 (1:1000, ab71548, Abcam), anti-collagen I (1:1000, BA2023, BOSTER), anti-SMA (1:1000, BA0002, BOSTER), anti-P62 (1:1000, ab56416, Abcam), anti-LC3B (1:1000, ab48394, Abcam), and anti-GAPDH (1:1000, BA2913, BOSTER). The densitometry of bands was performed by ImageJ software (version 1.46).