Ab106556

Total protein was isolated from cells using the RIPA (Radio-Immunoprecipitation Assay) lysis buffer. Nucleoprotein from cells was extracted using the Nucleoprotein Extraction Kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The concentration of all of the extracted protein samples was measured by the BCA Kit (Beyotime, Shanghai, China) according to the protocol. Protein samples were separated by SDS-PAGE (8% or 10% polyacrylamide gels) and waterishly transferred to polyvinylidene difluoride (PVDF) membranes. We blocked membranes with 5% nonfat milk for 1 h and then incubated overnight at 4 °C with specific primary antibodies against Rspo1 (1:1000 dilution, ab106556), Lgr4 (1:1000 dilution, ab137480), Histone H3 (1:1000 dilution, ab33309) from Abcam (Cambridge, MA, USA) and β-catenin (1:1000 dilution, D10A8), β-actin (1:1000 dilution, 13E5) from Cell Signaling Technology (San Antonio, TX, USA). After rinsing, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody for 1 h and visualized using the enhanced chemiluminescence detection system (Millipore, Billerica, MA, USA).

RASFs (8 × 10⁵) and serum samples were exposed to RIPA buffer to extract total proteins. Protein concentration was quantified as per the instruction of a bicinchoninic acid assay (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Afterward, the protein samples were loaded onto 10% separating gel and then shifted onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was incubated with the primary antibodies against proliferating cell nuclear antigen (PCNA) (ab29; 1:1000; Abcam, Cambridge, MA, USA), Cyclin D1 (ab16663; 1:200; Abcam), Cyclin E1 (ab33911; 1:1000; Abcam), BCL2-associated x protein (Bax) (ab32503; 1:5000; Abcam), B-cell lymphoma-2 (Bcl2) (ab32124; 1:1000; Abcam), IL-1β (ab216995; 1:1000; Abcam), TNF-α (ab183218; 1:1000; Abcam), RSPO1 (ab106556; 1:500; Abcam), and β-actin (ab8226, 1:1000; Abcam). Then, the membrane was incubated with HRP-conjugated secondary antibody (ab205718/ab205719; 1:5000; Abcam), and the protein bands were visualized by electrochemiluminescence. The intensities of protein bands were quantified using Image Lab analysis software (Bio-Rad, Hercules, CA, USA).