Immunohistochemical Scoring Protocol for Protein Expression

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.