In total, 5 × 106 CRC cells were harvested with a cell scraper, and RIPA lysis buffer (P0013B, Beyotime) was used to extract total protein. The concentration of total protein was measured with a bicinchoninic (BCA) protein assay kit (PC0020, Solarbio). Samples were mixed with 5x loading buffer and then heated at 100 °C for 10 min. Then, 25 μg of total protein was loaded onto SDS–PAGE gel, and a standard western blot procedure was performed. The separated proteins were transferred to PVDF membranes (IPVH00010, Millipore) and blocked with 5% nonfat milk. The membrane was incubated with primary antibodies overnight at 4 °C and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. The HRP-labeled proteins were visualized with an ECL kit (WBKLS0010, Millipore) and an automatic chemiluminescence image analysis system (5200 Multi, Tanon). All full-length uncropped western blots are provided in the Supplementary Material. The following antibodies were used: an anti-p-STAT3 antibody (Y705) (Cy6566, Abways), an anti-STAT3 antibody (sc-482, Santa Cruz), an anti-HuR rabbit polyclonal antibody (11910-1-AP, Proteintech), an anti-HuR mouse monoclonal antibody (mAb; 66549-1-Ig, Proteintech), an anti-β-actin antibody (30101ES60, Yeasen Biotech Co.), an anti-BCL2 antibody (12789-1-AP, Proteintech), an anti-C-MYC antibody (10828-1-AP, Proteintech), an anti-cyclin D1 antibody (sc-753, Santa Cruz), an anti-PARP antibody (#9542, CST), an anti-cleaved caspase-3 antibody (#9661, CST), an anti-caspase-3 antibody (#9662, CST), an anti-Flag antibody (F1804, Sigma), an anti-Ub (P4D1) mouse mAb (#3936, CST), an anti-K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb (#8081, CST), an anti-β-TrCP antibody (sc-390629, Santa Cruz), anti-rabbit IgG (sc-2027, Santa Cruz), anti-mouse IgG (sc-2025, Santa Cruz), peroxidase-conjugated goat anti-rabbit IgG (33101ES60, Yeasen Biotech Co.), and peroxidase-conjugated goat anti-mouse IgG (33201ES60, Yeasen Biotech Co.).
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