Radioimmunoprecipitation assay lysis solution

Total protein extraction from HT-1376 and HT-1197 cell lines was performed using a radioimmunoprecipitation assay lysis solution (Thermo Fisher Scientific, Inc.), and BCA assay was used for protein quantification. Subsequently, 20 µg protein from each sample was subjected to 10% SDS-PAGE gel electrophoresis, followed by gel transfer to polyvinylidene fluoride membranes. Membranes were blocked with 5% skimmed milk at room temperature for 2 h, followed by washing with PBS and incubation with primary antibodies including rabbit anti-human EGFR (1:2,000; cat. no. ab131498; Abcam, Cambridge, UK) and rabbit anti-human GAPDH (1:1,000; cat. no. ab8245; Abcam) overnight at 4°C. Following washing with PBS, the membranes were incubated with an anti-rabbit IgG-horseradish peroxidase secondary antibody (1:1,000; cat. no. MBS435036; MyBioSource, Inc., San Diego, CA, USA) at room temperature for 1 h. Then, enhanced chemiluminescent reagents (Sigma-Aldrich; Merck KGaA) were added to detect the signals. Membranes were scanned using MYECL™ Imager (Thermo Fisher Scientific, Inc.), and ImageJ v1.46 software (National Institutes of Health, Bethesda, MD, USA) was used to normalize the relative expression level of EGFR to the endogenous control GAPDH.

Radioimmunoprecipitation assay lysis solution (Thermo Fisher Scientific, Inc.) was used for protein extraction from CCD-25Lu and Hs 683 cells and protein concentrations were measured by BSA assay. Subsequently, 10% SDS-PAGE was performed with 25 µg denatured protein in each well. Following protein transfer to polyvinylidene difluoride membranes, blocking was performed with 5% skimmed milk at room temperature for 2 h. After three washes with TBS and 0.3% Tween (TBST) buffer, the membranes were incubated with rabbit anti-human primary antibodies against TGF-β1 (1:1,500; cat. no. ab92486; Abcam, Cambridge, UK) and GAPDH (1:1,200; cat. no. ab9485; Abcam) at 4°C overnight. The membranes were then washed three times with TBST buffer, followed by incubation with goat anti-rabbit IgG-horseradish peroxidase secondary antibody (1:1,000; cat. no. MBS435036; MyBioSource, Inc., San Diego, CA, USA) for 2 h at room temperature. Subsequently, the membranes were washed three times with TBST buffer and the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.) was used to develop signals. Signals were scanned using MYECL™ Imager (Thermo Fisher Scientific, Inc.). TGF-β1 expression was normalized to that of GAPDH using ImageJ 1.6 software (National Institutes of Health, Bethesda, MD, USA).