P70 s6k pt389

Western blotting was conducted as previously described [26 (link)]. In Western blotting, the following antibodies were used at the indicated dilutions: p70 S6K–pT389 (1:1000, #9234), p70 S6K (1:1000, #9202), phospho–CK2 substrate (1:1000, #8738) from Cell Signaling Technology (Danvers, MA, USA); CK2β (1:500, 22418-1-AP), NOLC1 (1:1000, 11815-1-AP), and β-actin (1:5000, 66009-1-Ig) from Proteintech Group (Chicago, IL, USA); TOP2A-pS1469 (1:1000, PA5-64536) from Affinity Biosciences (Cincinnati, OH, USA); CK2β–pS209 (1:1000, 44-1090G) from Invitrogen (Waltham, MA, USA); TOP2A (1:200, sc-365916), CK2α (1:1000, sc-373894) from Santa Cruz Biotechnology (Dallas, TX, USA); goat anti-mouse IgG-HRP (1:5000, 115-035-062), goat anti-rabbit IgG-HRP (1:5000, 111-035-003) from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

All human urothelial cell carcinoma cell lines were obtained from the American Type Culture Collection (Rockville, MD). The RT4 and T24 cells were cultured in McCoy's 5A medium (Sigma-Aldrich, St. Louis, MO). The E6 and TCCSUP were cultured in DMEM medium (GIBCO^® Grand Island), NY. The TSGH8301 and J82 were cultured in RPMI-1640 medium (GIBCO^® Grand Island). The grade of these bladder cell line and urothelial carcinoma cell lines: E6 (Normal), RT4 (Grade 1), TSGH8301 (Grade 2) and J82, T24 (Grade 3), respectively [38 (link)–41 (link)]. All media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin-G, 100 g/ml streptomycin, and 2 mM L-glutamine (CCS; HyClone, Logan, UT). All cell lines were incubated at 37°C with 5% CO₂. The primary antibodies used in this study included the following: mTOR-pS2481, mTOR-pS2448, mTOR, p70S6K-pT389, ERK, ERK-p, Gab1, AKT (Cell Signaling, Boston, MA), mSin1 (Millipore, Billerica, MA), p70S6K, AKT-pS473, c-myc (Santa Cruz Biotechnology Inc., Santa Cruz, CA), rictor, raptor and beta-actin (GeneTex, Hsinchu, Taiwan). EGF (100 ng/ml) and insulin (2 μg/ml) were purchased from PEPROTECH, and LY294002 (20 μM) was purchased from GIBCO^®.