Sc 56878

HEK293 was originally purchased from American Type Culture Collection (Manassas, VA, USA). Mouse embryonic fibroblasts (MEFs) were extracted from NIH mouse embryos as report [23 (link)]. Cells were cultured using Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 μg/ml), and at 37° C in 5% CO₂. Rapamycin (HY-10219) and Stat-3 specific inhibitor (HO-3837, HY-100453) were ordered from MedChemexpress (Monmouth Junction, NJ, USA). Primary antibodies against Runx-2 (sc-390351), OPN (sc-21742), p-Akt1/2/3 (sc-377556), Akt1/2/3 (sc-56878), p-mTOR (sc-517464), and mTOR (sc-293089) were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA); primary antibodies against IL-6 (21865-1-AP), Rictor (27248-1-AP), and Raptor (20984-1-AP) were ordered from Proteintech (Wuhan, China); primary antibody against β-actin (AC026) was ordered from ABclonal technology (Wuhan, China).

The extracted cell total protein was loaded, separated by 10% SDS-PAGE, and the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with TBST solution containing 5% nonfat milk at room temperature for 1 h. The membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-human monoclonal PCNA antibody (ab92552; 1:1000 dilution; Abcam, UK), rabbit anti-human monoclonal cleaced casepase-3 antibody (ab2302; 1:1000 dilution; Abcam, UK), mouse anti-human monoclonal antibody to phosphorylated protein kinase B (p-Akt; sc-81433; 1:1000 dilution; Santa Cruz Biotechnology, USA), mouse anti-human monoclonal Akt antibody (sc-56878; 1:1000 dilution; Santa Cruz Biotechnology, USA), and rabbit anti-human monoclonal β-actin antibody (ab179467; 1:1000 dilution; Santa Cruz Biotechnology, USA).The membranes was washed by TTBS for 5 times, and then followed by HRP-linked secondary antibodies (ab6721 and ab6789; 1:1000 dilution; Abcam, USA) for 2 h at room temperature, and the protein signals were detected using an enhanced chemiluminescence reagent (Bio-Rad Laboratories, USA).