Bicinchoninic protein assay kit

Total protein was extracted from the fibroblasts when they reached 60–80% confluence and protein concentrations were measured using a Bicinchoninic Protein Assay kit (Thermo Fisher Scientific, Inc.). A total of 10 µg protein was separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Following the blocking of non-specific binding with 5% non-fat milk dissolved in TBS + 0.05% Tween-20 at room temperature for 2 h, the membranes were incubated with the following antibodies: Anti-cleaved poly [ADP-ribose] polymerase [PARP; 1:1,000; catalog no. 5625; Cell Signaling Technology, Inc. (CST), Danvers, MA, USA], anti-GAPDH (1:1,000; catalog no. 8884; CST), anti-B cell lymphoma 2 (Bcl-2; 1:1,000; catalog no. 4223; CST), anti-Bax (1:1,000; catalog no. 5023; CST) and anti-mothers against decapentaplegic homolog 3 (Smad3; 1:1,000; catalog no. ab40854; Abcam, Cambridge, UK) at 4°C overnight. Following washing with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies (1:1,000; catalog no. 7074; CST) and the protein expressions were visualized using the Enhanced Chemiluminescence System (EMD Millipore, Billerica, MA, USA). The bands were quantified by densitometry using Image J 1.46r software (National Institutes of Health, Bethesda, MD, USA). All reactions were performed in triplicate.

The cells were lysed in radioimmunoprecipitation assay buffer with protease inhibitor (both Thermo Fisher Scientific, Inc.). The protein concentration was determined using a Bicinchoninic Protein Assay kit (Thermo Fisher Scientific, Inc.). Proteins (50 µg per lane) were separated on 10% SDS-PAGE, which were subsequently transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% non-fat milk in phosphate buffered saline with 0.1% Tween-20 at 4°C overnight. Subsequently, the PVDF membranes were incubated at room temperature with rabbit antibodies against SPRY4 (1:500; Abcam, Cambridge, UK; cat. no. ab176337) or GAPDH (1:500; Abcam; cat. no. ab9485) for 4 h, followed by incubation at room temperature with a goat anti-rabbit HRP-conjugated secondary antibody (1:5,000; cat. no. ab6721; Abcam) for 40 min. The signals on the membrane were examined using a Chemiluminescent Substrate kit (Thermo Fisher Scientific, Inc.). Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used to determine the protein expression.

Tumor cells were homogenized in lysate buffer containing protease-inhibitor (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and were centrifuged at 8,000 × g at 4°C for 10 min. The supernatant was used for analysis of the total protein using a bicinchoninic protein assay kit (Thermo Fisher Scientific, Inc.). Protein samples (20 µg) were separated on 15% sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA) as previously described (21 (link)). Protein was blocked with 5% bovine serum albumin reagent (BSA; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 1 h at 37°C. For western blotting, primary rabbit anti-human antibodies against PDGFR-β (cat. no. ab32570), RET (cat. no. ab134100) and β-actin (cat. no. ab8226) (all 1:500 dilution; Abcam, Cambridge, UK), were incubated overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated polyclonal anti-rabbit immunoglobulin (Ig) G antibody (1:10,000; cat. no. HAF008; R&D Systems, Inc., Minneapolis, MN, USA) for 2 h at room temperature. A Ventana Benchmark automated staining system was used for analyzing protein expression (Olympus BX51; Olympus; Tokyo, Japan).