Ps2448 mtor

Primary antibodies were obtained as follows: LC3B, tubulin, and MuRF1 antibodies were acquired from Novus Biologicals (Centennial, CO, USA), Abcam (Cambridge, UK), and Santa Cruz Biotechnology (Dallas, TX, USA), respectively; mTOR, pS2448-mTOR, Akt, pS473-Akt, ribosomal S6, pS235/236-ribosomal S6, 4EBP1, pS65-4EBP1, FoxO1, pS256-FoxO1, Atrogin-1, ULK1, pS757-ULK1, AMPK, and pT172-AMPK were obtained from Cell Signaling Technology (Danvers, MA, USA). All secondary antibodies were procured from Jackson Immuno Research Laboratories, Inc. (West Grove, PA, USA). The intercostal, diaphragm, and gastrocnemius muscles from male Sprague Dawley rats, either 6-month-old or 20-month-old were obtained from the Aging Tissue Bank (Pusan University, Pusan, Korea) [49 (link)].

Antibodies used included pS2448 mTOR, mTOR, pS235-S236 S6, S6 ribosomal subunit, pS473 Akt (Cell Signaling Technology). The rabbit anti-CD3ζ 448 antibody was produced in the laboratory of Dr. B. Alarcón (Centro de Biología Molecular “Severo Ochoa” (CBMSO), Madrid, Spain). Anti-α-Tubulin (clone DM1A, AB_477593), anti-α-Tubulin FITC-conjugated (clone DM1A, AB_476968) and anti-β-Actin (clone AC-15, AB_476692) from Sigma Aldrich; anti-CD3ϵ (clone HIT3a) from BioLegend; αCD3/αCD28 tetramers (Immunocult activator) were from StemCell Technologies; anti-CD28 (clone CD28.2), anti-P150-Glued (1/p150Glued (RUO), AB_397846), anti-CD3ϵ-APC and anti-CD4-APC from BD Biosciences. Isotype control–FITC and isotype control-APC were from Immunostep. Reagents and probes are as follows: MitoTracker™ Orange CMTMRos, CellTracker™ Blue CMAC Dye and Phalloidin Alexa Fluor™ 647 from LifeTechnologies (Invitrogen). Nonyl-acridine-orange was from SigmaAldrich.

Monoclonal antibodies raised against the following proteins were purchased from the indicated commercial sources: α-tubulin (Sigma-Aldrich, #T5168), Myc-tag (MBL, #M047-3), and GAPDH (MBL, #M171-3). Polyclonal antibodies raised against the following proteins were purchased from the indicated commercial sources: ARHGEF3 (Abcam, #ab154263), HHAT (ABGENT, #AP5503a for Figures 3C, 7D, 7F, and 7I; and Sigma, #SAB2105163 for Figure 7G-i), OCT-4A (Cell Signaling, #2840), SOX2 (Cell Signaling, #3579), NANOG (Cell Signaling, #4903), mTOR (Cell Signaling, #2983), mTOR-pS2448 (Cell Signaling, #5536), AKT (Cell Signaling, #75692), and AKT-pS308 (Cell Signaling, #13038).

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^®.