Ab25121

Proteins were extracted from liver tissues randomly from three individual rats in each group using RIPA. Samples were separated by SDS-PAGE and transferred to PVDF membrane. Subsequently, membranes were blocked and probed with primary antibodies followed by secondary horseradish peroxidase-conjugated antibody. Immunolabeled proteins were detected by incubation with ECL substrate and the gray density was measured. Target proteins levels were normalized against the level of β-actin or Lamin B1. The following antibodies were used: TLR4 (1:1000, ab22048, Abcam, United Kingdom), MyD88 (1:1000, #4283, Cell Signaling Technology, United States), TRAF6 (1:800, sc-7221, Santa Cruz, CA, United States), NF-κB p65 (1:2000, ab16502, Abcam, United Kingdom), TGF-β₁ (1:2000, ab25121, Abcam, United Kingdom), α-SMA (1:1000, ab5694, Abcam, United Kingdom), β-actin (1:1000, sc-47778, Santa Cruz, CA, United States), and Lamin B1 (1:10000, ab16048, Abcam, United Kingdom).

In situ TGF-β1 expression in the tracheal scar was assessed by immunohistochemistry (IHC) of the tracheal tissue sections used for microscopy with a rabbit polyclonal anti-TGF-β1 antibody (Abcam ab25121, USA) using the biotin-streptavidin-peroxidase system (Vectastain Universal Quick Kit, Burlingame, CA). The sections were incubated with 3,3′-diaminobenzidine (DAB, BioCare Medical, USA) and counterstained with CAT hematoxylin (BioCare Medical, USA). After completing the staining, TGF-β1 expression was quantified in the entire sample circumference using the program ImageJ (http://rsbweb.nih.gov/ij/), which was developed by the National Institutes of Health (NIH), and the plugin IHC Profiler [17 (link)].

IHC staining was performed on tissue microarrays as previously described (19 (link)). The following antibodies were used: anti–PLXND1 (ab28762; Abcam, Cambridge, MA) and anti–TGF-β1 (ab25121; Abcam, Cambridge, MA). Expression of PLXND1 were semiquantitatively classified according to the immunoreactive H-score (HS; range 0–300). Subsequently, stratification scoring was conducted according to H-score as follow: HS < 80, scored as 0; 80 < HS <120, scored as 1; 120 < HS < 200, scored as 2; HS > 200, scored as 3. If the score was equal to or greater than 2, the tumor was considered to have high PLXND1 expression; otherwise, low PLXND1 expression was classified. The expression levels of target proteins in tissue were examined by two independent pathologists blinded to the clinical characteristics of the patients.

IHC was performed as previously described [24 (link), 25 (link)]. Antibody against SHCBP1 (12672–1-AP) was purchased from Proteintech (Wuhan, China). Antibodies against phosphorylated (p)-Smad2 (ab53100), p-Smad3 (ab51177) and TGF-β1 (ab25121) were purchased from Abcam (Cambridge, MA). All sections were assessed and scored by two independent pathologists (Xiaoying Wang and Chengjun Zhou) in a blind manner. Ten high-power fields were selected randomly for each slide. The expression levels of each marker in tumor cells were independently evaluated. The percentage of positive-staining cells were graded on a scale of 0 to 3, with less than 5% positive-staining cells as grade 0, 5 to 25% as grade 1, 26 to 50% as grade 2, and more than 50% as grade 3. The intensity of staining was also graded on a scale of 0 to 2, with negative to weak intensity as grade 0, weak to moderate intensity as grade 1, and moderate to strong intensity as grade 2. After that, the percentage score was multiplied by the intensity score. A final score between 0 and 2 was defined as low expression, and a score higher than 2 was defined as high expression.