Bs0638r

After drying naturally, BCPAP cells were immersed in 4% paraformaldehyde fixation solution to improve the cell permeability. Then, BCPAP was coincubated with anti-TβR1 (bs0638R, Bioss, China) and anti-p-Smad3 (ab52903, Abcam, UK) with a dilution rate of 1 : 100 for 2 hours. Followed by exposing to fluorescent dye-conjugated secondary antibody (TRITC) diluted at 1 : 100, BCPAP was mounted with DAPI. The expression of target protein was observed and recorded by a laser scanning confocal microscope (710, Zeiss, Germany).

The total protein was obtained using extraction kit (KGP250, KeyGen BioTECH, Nanjing, China), followed by the quantification using BCA protein concentration detection kit (KGA902, KeyGen BioTECH, Nanjing, China). By applying gel preparation kit (KGP113, KeyGen BioTECH, Nanjing, China), sodium dodecyl sulfate- (SDS-) PAGE was conducted followed by the membrane transfer. Thereafter, WB was performed, using anti-NIS (24324-1-AP, Proteintech Group, Inc., China), anti-Rap1GAP (ab32373, Abcam, UK), anti-TGF-β1 (ab215715, Abcam, UK), anti-Foxp3 (bs10211R, Bioss, China), anti-TβR1 (bs0638R, Bioss, China), anti-p-Smad3 (ab52903, Abcam, UK), anti-Smad3 (ab40854, Abcam, UK), anti-bcl-2 (ab182858, Abcam, UK), and anti-Bax (ab182733, Abcam, UK) with dilution rates of 1 : 1000, 1 : 10000, 1 : 1000, 1 : 1000, 1 : 1000, 1 : 2000, 1 : 1000, 1 : 2000, and 1 : 2000, respectively. After incubation with the secondary antibody, the membrane was colorized and imaged using G: BOX chemiXR5 (syngene, UK).

Cells were fixed in 4% paraformaldehyde (30 min), permeabilized with 0.1% Triton X-100 (15 min), blocked using 3% BSA (30 min), incubated with primary antibodies at 4 °C (12 h), and then stained with AF 647- or AF 488-labeled secondary antibodies (Thermo, USA). The primary antibodies used in the study were anti-Ki67 (bs-23105R, Bioss, China), anti-α-SMA (bsm-33188M, Bioss, China), anti-fibronectin (anti-Fn; bs-0666R, Bioss, China), anti-transforming growth factor-β1 (anti-TGF-β1; bsm33345M, Bioss, China), anti-transforming growth factor-β2 (anti-TGF-β2; bs-20412R, Bioss, China), anti-transforming growth factor-β3 (anti-TGF-β3; AF8142, Beyotime, China), anti-transforming growth factor-β receptor type I (anti-TGF-βR1; bs0638R, Bioss, China), and anti-transforming growth factor-β receptor type II (anti-TGF-βR2; AF8151, Beyotime, China). Nuclear staining was performed with DAPI (Beyotime, China). Images were examined under a fluorescence microscope (BX63, Olympus, Japan).