HIR cells were serum starved in DMEM for 4 h at 37°C. After the cells were stimulated with insulin or aptamers for 15 min, the medium was immediately removed and the cells were washed out with ice-cold phosphate buffered saline (PBS) buffer. The cells were lysed in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 mM -glycerophosphate, 50 mM NaF, 1.5 mM sodium orthovanadate, and 1% Triton-X. Cell debris was removed by centrifugation for 15 min at at 4°C, and the protein concentration of the supernatants was measured using a BCA protein assay kit (Pierce, Rockford, Illinois). The cell lysates were boiled for 10 min at 95°C in the sample buffer and were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were transferred to a nitrocellulose membrane, and the membrane was blocked in TTBS buffer containing 10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.05% Tween 20, and 5% nonfat dry milk. After an overnight incubation with specific primary antibodies at 4°C and incubation with IRDye800-conjugated anti-rabbit or anti-mouse IgG secondary antibodies for 1 h at room temperature, the blots were visualized by an Odessay infrared imaging system (LI-COR Bioscience, Lincoln, Nebraska). Anti-IR antibody, anti-phospho IR (pY1150/pY1151) antibody, and anti-phospho IRS1 (pY632) antibody were purchased from Santa Cruz Biotechnology (Dallas, Texas). Anti-phospho AKT (pS473) was purchased from Cell Signaling Technology (Danvers, Massachusetts).