Western blotting

BaF3 cells from each group were washed with PBS, resuspended and centrifuged. The supernatant was collected and radioimmunoprecipitation lysis buffer was added (cat. no. P0013B; Beyotime Institute of Biotechnology, Haimen, China) and 0.1 mM phenylmethylsulfonyl fluoride. Following gentle shaking and resuspension, the cells were incubated on ice for 30 min and centrifuged for 10 min (241 × g; 4°C). The total cellular protein, which remained in the supernatant, was subsequently collected. The protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology) and the protein concentration was adjusted to 4 µg/µl. Total cellular protein (30 µg) was extracted for 10% SDS-PAGE, followed by transferring to the nitrocellulose (NC) membranes by 50 V wet electrotransfer and blocked in 5% skim milk [TBS with Tween-20 (TBST) formulation] for 1.5 h. at room temperature. Antibodies were diluted in primary antibody diluents, according to the manufacturer's protocol. Primary antibodies included CRLF2 (rabbit anti-human; 1 µg/ml; cat. no. ab109626; Abcam, Cambridge, UK), phosphorylated (p)-AKT (rabbit anti-human; 1:1,000; cat. no. ab52192; Abcam), p-mTOR (rabbit anti-human; 1:1,000; cat. no. ab109268; Abcam), eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1; rabbit anti-human; 1:5,000; cat. no. ab32024; Abcam), ribosomal protein S6 kinase β-1 (S6K1; rabbit anti-human; 1:5,000; cat. no. ab32529; Abcam), and GAPDH (rabbit anti-human; 1:10,000; cat. no. ab181602; Abcam). The blocked NC membranes were placed in a plastic container into which the above antibodies were added, followed by agitation and storage at 4°C for 24 h. The membranes were washed in TBST three times (15 min/wash) and were subsequently incubated at room temperature for 2 h following the addition of diluted HRP-labeled secondary antibodies IgG (goat anti-rabbit; 1:5,000; cat. no. ab205718; Abcam). The membranes were washed in TBST three times (15 min/wash) and treated with enhanced chemiluminescence (ECL western blotting substrate kit; Thermo Fisher Scientific, Inc.). Samples were photographed using SmartView Pro 2000 (UVCI-2100; Major Science, Saratoga, CA, USA). Protein banding grayscale analysis was performed using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).