Radioimmunoprecipitation assay kit

Total protein was extracted using a radioimmunoprecipitation assay kit (Beyotime, Shanghai, China), and protein concentration was determined using a BCA kit (Beyotime). Equal amounts of protein were separated by loading onto 10% SDS-PAGE, then the proteins were transferred to polyvinylidene difluoride membranes, blocked with 5% nonfat milk for 1 h, mixed with primary antibodies anti-YAP1 (ab52771; 1:1000, Abcam) and anti-GAPDH (ab8245; 1:1000, Abcam) overnight at 4 °C, and combined with goat anti-Rabbit IgG-HRP (ab205718; 1:2000, Abcam) for 2 h. Protein bands were colorized with enhanced chemiluminescence reagent (Beyotime) and evaluated by Image J software.

Proteins were extracted from cells and tissues using a radioimmunoprecipitation assay kit (Beyotime, Shanghai, China) supplemented with 0.1% protease inhibitors and 0.1% phosphorylase inhibitors. The protein concentration was quantified using the BCA Protein Assay Kit (Beyotime, Shanghai, China). Subsequently, the protein samples were separated on 10% SDS-PAGE gels, and the separated proteins were transferred to a PVDF membrane (Millipore). The membranes were incubated in 5% milk for 2 h at room temperature to block non-specific binding, and then incubated with appropriate antibodies overnight at 4°C. The following primary antibodies were used: ASRGL1 (#11400-1-AP, 1:1,000, Proteintech), GAPDH (#5174, 1:1,000, CST). The membranes were visualized with an ECL detection system after incubation with secondary antibody at room temperature for 2 h.

Western blot assays were performed as previously described^{34 (link)}. Briefly, the total cellular proteins were extracted by using reagents in a Radio Immunoprecipitation Assay kit (Beyotime, Shanghai, China). The extracted proteins were then separated by SDS-PAGE, and the individual protein bands were transferred onto a PVDF membrane, which was subsequently blocked with 5% skim milk. The membrane was then incubated with primary antibodies against E-cadherin (1:800, ab76055, Abcam, Cambridge, MA, USA), FN (1:5000, ab45688, Abcam, USA), β-catenin (1:8000, ab32572, Abcam, USA), α-SMA (1:1000, ab28052, Abcam, USA), LC3B (1 μg/mL, ab48394, Abcam, USA), p62 (1:2000, ab155686, Abcam, USA), or the internal reference GAPDH (1:20000, ab128915, Abcam, USA). Next the membrane was incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies. An enhanced chemiluminescence detection system was used to detect areas of luminescence, and the relative-staining intensity of each protein band was quantitated using Image Plus Pro software (Ver. 6.0, Media Cybernetics, Inc., Rockville, MD, USA).