Super ecl kit

WB was performed as described earlier [46 (link)]. NRCFs were lysed in radio-immunoprecipitation assay buffer (RIPA; Cell Signaling Technology, Danvers, MA, USA) containing protease inhibitor (Amresco, Solon, OH, USA) and homogenized with sonication. Samples were centrifuged for 10 min at 12,000 rcf at 4 °C. Protein concentration in the samples was measured using a bicinchoninic acid (BCA) assay kit (Thermo Scientific, Waltham, MA, USA). For the analysis of isolated mEVs, isolates were mixed with 10× RIPA buffer in 1:10 dilution right before use. For the myofibroblast transformation assay, 6 µg of protein was used, and for mEV analysis, 30 µL of each sample was used. Antibodies are presented in Supplementary Table S2. For control, Vimentin was used as a common fibroblast marker. Signals were visualized using enhanced chemiluminescence kit (ECL, Bio-Rad, Hercules, CA, USA) or super ECL kit (Bio-Rad, Hercules, CA, USA) by Chemidoc XRS+ (Bio-Rad, Hercules, CA, USA). Band intensities were measured using Image Lab software (Bio-Rad, Hercules, CA, USA). α-SMA and Vimentin were normalized to DDR2, and TGF-β was normalized to GAPDH.

Total proteins were extracted using the Western and IP cell lysis kit (Beyotime, Songjiang, Shanghai, China) according to the manufacturer’s instructions and the protein concentrations were determined using the BCA protein assay kit (Beyotime, Songjiang, Shanghai, China). Total proteins (20 mg per lane) were separated on 10% SDS-PAGE gels and transferred onto PVDF membranes (Cat. No. 88518, Thermo Fisher, Waltham, MA, USA). The membranes were then blocked in 5% skim milk for 2 h at room temperature before being incubated with primary antibodies against ITGB3, FAK, pFAK, MMP2, MMP9, Bcl-2, Bax, and GAPDH (ITGB3, FAK, pFAK, MMP2, MMP9, Bcl-2 and Bax primary antibodies were diluted at 1:1000, GAPDH primary antibody was diluted at 1:2000) for overnight at 4°C. The membranes were then rinsed three times with TBS-T (Tris buffered saline with 0.1% Tween-20) and incubated with a secondary antibody solution for 2 h at room temperature. The secondary antibody was diluted at 1:10000 in skim milk. Protein bands were visualized using a new super ECL kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. The amount of protein in each band was quantified by software Quantity One 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All assays were repeated three times in parallel.