Polyvinylidene difluoride membrane 0.2 μm

For protein isolation, cells were lysed using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with 1 mM PMSF (Beyotime Biotechnology, Shanghai, China). The extracted total proteins were then separated using 12.5% SDS-PAGE and subsequently transferred to a polyvinylidene difluoride membrane (0.2 μm; Millipore, Burlington, MA, USA) utilizing a semi-dry transfer system (Bio-Rad; Hercules, CA, USA). The membrane was blocked with a rapid blocking solution (YaMei, Hangzhou, China) for 12 min. After blocking, the membranes were incubated overnight with primary antibodies at 4 °C. Subsequently, the membranes were washed four times with TBST, each wash lasting 9 min. The membranes were then incubated with appropriate secondary antibodies for 1 h at room temperature, followed by four additional 9 min washes with TBST. For detection, the membrane was incubated with Enhanced Chemiluminescence (ECL) detection reagent (Thermo Scientific, Rockford, IL, USA) and imaged using a chemiluminescence detector (Tanon, Shanghai, China). Protein bands were scanned in grayscale using a Tanon Gel Imaging System (Tanon, Shanghai, China). Protein levels were quantified using ImageJ software (1.8.0), with each protein normalized against vinculin levels. The specific antibodies utilized are detailed in Table 2.

Samples containing equal amounts of protein were subjected to 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (0.2 μm; Millipore, USA). After being blocked with 2% bovine serum albumin, the blots were probed with anti-TGFBR1, anti-TGFBR2, anti-IGF1 and anti-IGF1R antibody (Abcam), and anti-GAPDH antibody (ZhongShanJinQiao). After being washed with tris-buffered saline and Tween 20, the membranes were treated with horseradish peroxidase-conjugated secondary antibody (ZhongShanJinQiao) at room temperature for 1 h and visualized by enhanced chemiluminescence (Millipore, USA).