Anti ps6k

CD8⁺ T cells were either left untreated or stimulated with CD3/CD8 mAbs or OT-I tetramers. The inhibitors AKT V (1 μM) AKT-VIII (2 μM) AKT XII (5 μM;all Calbiochem) or Rapamycin (2 mM) were directly added to the samples after stimulation. Anti-AMPKα, anti-phospho(p)AMPKα (Thr172), Anti-p-ERK1/2, anti-pS6K (all Cell Signaling) and anti-β-actin (clone AC-15) (Sigma-Aldrich) were used for Western blotting.

Erlotinib was kindly provided by F. Hoffman-La Roche Ltd; TPX-0005 was from TP Therapeutics, Inc.; dasatinib was purchased from Bio Vision; sorafenib was from Toronto Research Chemicals Inc; the other inhibitors were from Selleck Chemicals. Anti-HER2 and anti-pHER2 antibodies were purchased from Upstate Biotechnology; anti-EGFR, anti-pEGFR, anti-pHER3, anti-MET anti-pMET, anti-ERK1/2, anti-pERK1/2, anti-AKT, anti-pAKT (T308 or S473), anti-STAT3, anti-pSTAT3, anti-PTEN, anti-AXL, anti-CDCP1, anti-pCDCP1, anti-SRC, anti-FYN, anti-LYN, anti-YES, anti-LCK, anti-pSFK (Y416), anti-β-catenin, anti-S6K, and anti-pS6K antibodies were from Cell Signaling Technology; anti-E-cadherin antibody was from BD biosciences; anti-HER3 antibody was from Santa Cruz Biotechnology; anti-β-actin antibody was from Abcam, Inc.; Anti-GAPDH antibody was from PROMEGA; α-tubulin antibody was sourced from Sigma-Aldrich.

Western blot analyses were performed as previously described^{26 ,28 (link)}. Briefly, cells were lysed and sonicated in RIPA buffer (Thermo Scientific, IL, USA). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoretic separation, protein bands were transferred to Millipore Immobilon-P membrane followed by immunoblotting with antibodies against molecules of interest. The following primary antibodies were used for immunoblotting: anti-Smad2 (Cell Signaling, MA, USA, 1:1000), anti-p-Smad2 (Cell Signaling, 1:1000), anti-Smad3 (Cell Signaling, 1:1000), anti-p-Smad3 (Cell Signaling, 1:1000), anti-Akt (Cell Signaling, 1:1000), anti-p-Akt (Thr308 and Ser473) (Cell Signaling, 1:1000), anti-S6K (Cell Signaling, 1:1000), anti-p-S6K (Cell Signaling, 1:1000), anti-α-SMA (SIGMA, 1:2000), anti-GAPDH (Santa cruz Biotechnology, CA, USA, 1:5000) and anti-HSP70 (Invitrogen, 1:1000) antibodies. Chemiluminescence detection was performed using the ECL reagent (Bio-Rad Laboratories. Inc., CA, USA). Signal intensities of bands were quantified with Image J software (NIH).

The aortas isolated from mice were embedded in optimum cutting temperature compound. Frozen sections (5 μm thick) were fixed and blocked with 5% goat serum. For double-immunofluorescent staining, the sections were stained for anti-mouse CD31 (1:100; BD Biosciences) and anti-LC3 (1:500; MBL), anti-ATG13 (1:200; Sigma-Aldrich) or anti-pS6K (1:100; Cell Signaling) antibodies, followed by incubation with fluorescent secondary antibodies (1:200; Invitrogen). Nuclei were counterstained with DAPI. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was used to evaluate cell apoptosis with the In Situ Cell Death Detection kit (Roche Diagnostics). Images were captured with a fluorescence microscope (BX61, Olympus) and images were analyzed using Image J software. The intensities of immunofluorescence staining images were analyzed by AlphaImager 2200 software.

Parotid glands were excised and homogenized in RIPA buffer with 5 mM sodium orthovanadate (Fisher Scientific), protease inhibitor cocktail (Sigma-Aldrich) and 100 mM PMSF (Fisher Scientific). The samples were then boiled for 10 minutes and sonicated until homogenous. 100 mg of each protein sample was added to 12% polyacrylamide gels and transferred to 0.45 µm Immobilon-P membranes (Millipore Billerica, MA). Membranes were blocked using either non-fat dry milk or 5% BSA then immunoblotted with one of the following antibodies: anti-ERK (Promega Madison, WI), anti-pAkt (Cell Signaling) and anti-pS6K (Cell Signaling). ECL substrate (Fisher Scientific) was used as instructed by the manufacturer for detection. The membranes were stripped using Restore Western Blotting Stripping buffer (Fisher Scientific) and then blocked and re-probed as described above.