Ab184948

C666-1 cells were lysed in cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.). Protein concentration was determined using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Protein (50 µg) was separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, Inc.). Subsequently, the membrane was blocked in 5% non-fat dried milk in Dulbecco's phosphate-buffered saline (DPBS; Thermo Fisher Scientific, Inc.) for 3 h at room temperature. The PVDF membrane was then incubated with rabbit anti-TGFβR2 monoclonal antibody (1:500; ab184948; Abcam, Cambridge, MA, USA), or rabbit anti-GAPDH monoclonal antibody (1:250; ab181602; Abcam) as an internal reference, for 3 h at room temperature. Subsequently, the membrane was washed with DPBS for 10 min and then incubated with mouse anti-rabbit secondary antibody (1:5,000; ab99697; Abcam) for 1 h at room temperature. Following washing with DPBS for 15 min, the immune complexes on the PVDF membrane were detected using an enhanced chemiluminescence western blotting kit (Pierce; Thermo Fisher Scientific, Inc.). Image-Pro Plus v. 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to analyze relative protein expression levels, represented as the density ratio vs. GAPDH.