Protein g sepharose

Digoxygenin-O-methylcarbonyl-ε-aminocaproic acid N-hydroxysuccinimide (DIG) ester (#11333054001), anti-DIG-POD Fab fragments (#11207733910) and cOmplete Mini EDTA-free protease inhibitor tablets (#1697498) were from Roche; Coomassie Protein Assay reagent from Thermo Scientific, polyethylenimine (PEI) from Polysciences Inc; Protein G-Sepharose, glutathione-Sepharose and enhanced chemiluminescence Western blotting kit (RPN2106) were from Amersham Bioscience; Precast NuPAGE® polyacrylamide Bis-Tris gels, Lipofectamine® 2000 transfection reagent, hygromycin, blasticidin, zeocin, puromycin, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (D1306) and Alexa Fluor® Phalloidin (A12381) were from Invitrogen; sequencing-grade trypsin from Promega; and microcystin-LR was purchased from Professor Linda Lawton (Robert Gordon University, Aberdeen, U.K.).

Antibodies to mTOR, rictor, raptor, mLST8, p18, phosphoThr1135 rictor, phospho-Ser240/244 S6 ribosomal protein, total S6 ribosomal protein, phospho-Thr389 p70 S6 kinase 1, phospho-Ser65 and phospho-Thr37/46 4E-BP1, p-Ser757 ULK1, phospho-Ser473 Akt, phospho-Thr308 Akt and GAPDH were from Cell Signaling Technology. Monoclonal anti-FLAGM2-Peroxidase (HRP) antibody, Flag M2 antibody, anti-FLAGM2 Affinity Gel, anti-HA and dimethyl pimelimidate were from Sigma Aldrich; PP6 subunit antibodies (SAPS1, SAPS2, SAPS3 and ANKRD28) from Bethyl laboratories; antibodies to ATP6V1B2, ATP6V0A2 and LAMP2 from Abcam; antibody to ATP6V1A was from GeneTex; polyethylenimine (PEI) from Polysciences; Protein G-Sepharose, glutathione-Sepharose and enhanced chemiluminescence Western blotting kit were from Amersham Bioscience; [³²P-γ]ATP was from Perkin Elmer; Protein G-Sepharose and immobilized glutathione from Pierce; Precast NuPAGE polyacrylamide Bis-Tris gels, Colloidal Coomassie, LDS sample buffer and Dextran Oregon Green 514 (D-7176) were from Invitrogen; dialyzed Fetal Bovine Serum (Cat # 26400036) was from ThermoFisher Scientific; sequencing-grade trypsin and CellTiter-Glo was from Promega; and microcystin-LR was purchased from Dr Linda Lawton (Robert Gordon University, Aberdeen, UK).