HCC tissues and adjacent normal tissues were homogenized in a lysate buffer containing protease-inhibitor (P3480; Merck KGaA, Darmstadt, Germany) and were centrifuged at 6,000 × g at room tempreture for 10 min. Western blot analysis was subsequently performed as previously described (20 (link)). Protein concentration was measured by a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Protein samples (10 µg/lane) were resolved by 12.5% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Merck KGaA). After blocking with 2% bovine serum albumin (Sigma-Aldrich; Merck KGaA), rabbit anti-human AKT1 (ab81283) or GAPDH (ab9485) antibodies (all, 1:2,000; Abcam, Shanghai, China) were incubated with protein samples for 2 h at room temperature. Membranes were washed with PBS for 15 min at room temperature and then incubation with horseradish peroxidase-conjugated polyclonal anti-rabbit immunoglobulin G antibodies (1:10,000; PV-6001; OriGene Technologies, Inc., Beijing, China) for 1 h at room temperature. Signals were visualized by chemiluminescence detection (Z370398; Merck KGaA). Densitometric quantification of the immunoblot data was performed using Quantity-One software (v3.24; Bio-Rad Laboratories, Inc., Hercules, CA, USA).