Mtor clone 7c10

The adherent and sphere-forming cells derived from CTBp and CNMp cells were collected by centrifugation and washed with phosphate-buffer saline. The cells were lysed in lysis buffer (Promega, Tokyo, Japan) with a protein inhibitor cocktail for 15 min. Approximately 10 μg of the extracted protein was analyzed with the following specific monoclonal antibodies against mTOR (clone 7C10, Cell signaling Technology, Tokyo, Japan), phospho-mTOR (Ser2448) (clone 49F9, Cell Signaling Technology), 4E-BP (clone 53H11, Cell Signaling Technology) and phospho-4E-BP (Thr37/46) (clone 236B4, Cell Signaling Technology), and polyclonal antibody against β-actin (Santa Cruz Biotechnology). The membranes were incubated with horseradish peroxidase-conjugated immunoglobulin G (IgG) (GE Healthcare, Tokyo, Japan). The immunoreactivity was detected using an ATTO EzWestLumi plus reagent (ATTO, Tokyo, Japan) and ImageQuant LAS4000 mini (GE Healthcare).

Exponentially growing cells were treated with 10 μM 8-Cl-Ado for various amounts of time and protein lysates were isolated and analyzed using an Odyssey Infrared Imaging System (LI-COR Biosciences) as described [19 (link)]. Primary antibodies were rabbit polyclonal antibodies against p-AMPKα (Thr172), AMPKa, p-acetyl-coA carboxylase (ACC) (Ser79), p-raptor (Ser792), p-mTOR (Ser2481), p4E-BP1 (Ser65) (Cell Signaling Technology), LC3B, beclin 1 (Novus Biologicals, Inc, Littleton, CO), p62 (Enzo Life Sciences, Farmingdale, NY); rabbit monoclonal antibodies against p-ULK1 (Ser555) clone D1H4, raptor clone 24C12, mTOR clone 7C10 (Cell Signaling Technology), ATG7 (Novus Biologicals, Inc, Littleton, CO); mouse monoclonal antibodies GAPDH clone 6C6 (Abcam, Inc, Cambridge, MA); and goat polyclonal antibody 4E-BP1 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA).

For cell protein extraction, samples were homoge-nized in RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, and 0.1% SDS). The protein concentration in each sample was estimated by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Immunoblotting was performed according to standard procedures. Antibodies used in this study include polyclonal antibodies against AT1R Cruz Biotechnology, Santa Cruz, CA, USA), p-mTOR (Ser2448, Clone 49F9, Cell Signaling Technology, Boston, MA, USA), mTOR (Clone 7C10, Cell Signaling Technology, Boston, MA, USA), β-actin (Sigma Aldrich, St Louis, MO, USA), and α-tubulin (GENE TEX). The first antibodies were detected by incubation with secondary antibodies conjugated to HRP (Bio/Can Scientific, Mississauga, ON, Canada) and developed using Western Lighting Reagent. The proteins were explored by X-ray films.