Phospho mtor ser2448 clone 49f9

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).

IHC staining was performed on freshly cut sections. Primary antibodies against HER2 (polyclonal, Dako, Glostrup, Denmark), EGFR (clone 31G7, Invitrogen, Carlsbad, CA, USA), phospho-AKT 1/2/3 (Thr308)-R (polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PTEN (clone 6h2.1, Dako, Denmark), phospho-mTOR (Ser2448) (clone 49F9, Cell Signaling Technology, Danvers, MA, USA), Ki67 (clone MIB1, Dako, Denmark), E-cadherin (#610181, BD Transduction Laboratories, San Jose, CA, USA), and beta-catenin (#610153, BD Transduction Laboratories, San Jose, CA, USA) were used. IHC stains were performed on a Bond automated stainer (Dako).