Tgf β1 ab215715

Antibodies were purchased from the following companies: Smad‐2/3(D7G7), α‐SMA (#19245), vimentin (#5741) and β‐actin (#P60709) were purchased from Cell Signalling Technology; TGF‐β1 (ab215715) was purchased from Abcam; NF‐κB (10745‐1‐AP) and GAPDH (10494‐1‐AP) were obtained from Pro‐teintech; CHRFAM7A (860647) was purchased from ZEN BIO; Kidney injury molecule 1(KIM‐1) (KCA0319031) antibodies was purchased from R&D system. Recombinant human TGF‐β1 were obtained from Pepro Tech (121809). Lipofectamine 2000 was purchased from Thermo Fisher (11668‐019) and the reverse transcription cDNA synthesis system and SYBR Green were obtained from TIANGEN.

Western blotting was performed using standard procedures. Total protein was isolated from tracheal tissues using radioimmunoprecipitation assay buffer (high)‐ phenylmethylsulfonyl fluoride (RIPA‐PMSF; SolarBio). Subsequently, total protein content was determined using a BCA assay kit (SolarBio). Equal amounts of protein were electrophoresed by SDS‐PAGE and then transferred to nitrocellulose membranes (Merck Millipore). Five percent skim milk was inhibited for 1 h and incubated for 12 h at 4°C with the following primary antibodies: COL1A1 (ab260043), CD4 (ab133616), CD25 (ab231441), and TGF‐β1 (ab215715), all antibodies were purchased from Abcam at a dilution of 1:1000. After three washes in TBST, they were incubated with the corresponding secondary antibodies for 1 h at room temperature. Finally, immunoblots were detected using an electrochemiluminescence plus reagent kit (Biosharp), and densitometric analysis of each target protein band was performed using ImageJ software (NIH).