Il 11

SMMC-7721 cells transfected with miR-23b mimics, mimic NC, inhibitor, and inhibitor NC were lysed by radioimmunoprecipitation assay buffer (RIPA) buffer [0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1 mM MgCl₂, 10 mM Tris-HCl, pH 7.4], and then disrupted with an ultrasonic cell disruptor on ice. After high-speed centrifugation, the debris was removed and the supernatant was collected. The total protein concentration was quantified using a protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA), as per the manufacturer's instructions. Proteins (50 µg) were separated on 10% SDS-polyacrylamide gels, and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). Non-specific sites were blocked by immersing the membranes in 5% non-fat dry milk (w/v) for 2 h at room temperature. Membranes were incubated overnight with primary antibodies against IL-11 (1:500; Santa Cruz Biotechnology, Inc.) or IL-11Rα (1:500; Santa Cruz Biotechnology, Inc.) at 4°C. After washing in Tris-buffered saline, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. GAPDH was used as an internal control, and signals were detected by enhanced chemiluminescent reagents.