Ripa lysate strong

Total protein and nucleoprotein of the EVs, cells or tumor tissues were extracted using RIPA lysate (strong) (Beyotime, Nanjing, China) and Nuclear and Cytoplasmic Protein Extraction kit (Beyotime). The concentration of proteins was evaluated using the BCA assay kit (Beyotime). Then, 30 μg total protein of each well was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% separating gel) and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin (BSA) and cultured with the primary antibodies: rabbit anti-β-catenin (ab32572, 1/5000, 92KDa), rabbit anti-NRSN2 (ab237739, 1/1000, 22KDa), rabbit anti-CD9 (ab92726, 1/2000, 25KDa), mouse anti-HSP70 (ab2787, 1/1000, 70KDa), rabbit anti-Calnexin (ab22595, 1 µg/mL, 90KDa), rabbit anti-Lamin B1 (ab16048, 0.1 µg/mL, 68KDa) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab181602, 1/10,000, 36KDa). Afterwards, the membranes were cultured with the secondary antibodies goat anti-mouse immunoglobulin G (IgG; ZB5305) and goat anti-rabbit (ZB-5301, 1/5000, ZSGB-Bio Co., Ltd, Beijing, China). The gray value of the target band was analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Expression of BDKRB1, THBS1, calcium pathway-related proteins (CaMKIIα/δ), and TGF-β pathway-related proteins (TGF-β and p-Smad3) was detected by western blotting in lung tissues of Sham, Model and Treatment groups. Briefly, total proteins were extracted from lung tissues using RIPA lysate (strong) (Beyotime, CN), and the concentration of total proteins was determined by a NanoDrop spectrophotometer (Thermo Fisher). An equal amount of total protein sample was taken to perform SDS-PAGE electrophoresis, followed by transfer onto a PVDF membrane. After 1 h block with 1% skimmed milk powder (Beyotime), PVDF membranes were incubated with primary and secondary antibodies for the target proteins, respectively. Details on the antibodies are available in Supplementary Table 1. Subsequently, PVDF membranes were color developed using BeyoECL Plus Kit (Beyotime), and images were collected by ChemiDoc MP chemiluminescent gel imaging system (Bio-Rad, Hercules, CA, USA). Grayscale values in gel blot were analyzed using Image J software (V.1.0; NIH, Bethesda, MD, USA).

Total protein was extracted with RIPA lysate (strong) (Beyotime Biotechnology
Co., Ltd, Shanghai, China). Then the concentration of proteins was tested by the
bicinchoninic acid assay (Beyotime). Next, the proteins were separated by
electrophpresis and transferred onto polyvinylidene difluoride membranes. The
membranes were blocked with 5% skimmed milk and washed by tris buffered saline
tween (TBST). After that, the membranes were cultured with the primary
antibodies at 4°C overnight: P21 activated kinase 2 (1:5000, ab76293, Abcam,
Cambridge, MA, USA), B-cell lymphoma 2 (Bcl-2) (1:1000, ab32124, Abcam),
Bcl-2-associated X protein (Bax) (1:2000, ab32503, Abcam), cleaved caspase-9
(1:2000, ab2324, Abcam), cleaved caspase-3 (1:2000, ab2302, Abcam) and β-actin
(1:5000, ab179467, Abcam). After being washed by TBST (3 times/10 minutes), the
membranes were cultured with secondary antibody horseradish
peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) H&L (1:2000,
ab205718, Abcam) for 1 hour and then washed by TBST (3 times/10 minutes) before
chemiluminescence developing and visualization. The image of protein blotting
was analyzed by Image J2x v2.1.4.7 software (Rawak Software, Inc. Germany).