Phospho mtor

The following antibodies were used: EGFR (sc-03-G), Akt1 (sc-1618), C14ORF37 (UT2) (sc-139226), Rictor (sc-271081), Raptor (sc-81537) pAMPK (S485/491), AMPK (sc-25793), Alpha-Tubulin (sc-5546), were purchased from Santa Cruz Biotechnology. pEGFR (Y1173) from Invitrogen, Myc-tag (Cat#2278S), mTOR (Cat#2972), phospho p70S6K1 (T389) (Cat#9204), pPKC (T514) (Cat#9379), pAkt (S243) (Cat#4060), phospho MAPK (Cat#9101S), MAPK (Cat#4695), ULK1 (Cat#8054), pULK1-S757 (Cat#6888), and pULK1-(Cat#5869) were purchased from Cell Signaling. Actin (Cat#A2228) was from Sigma. PKC (Cat# ab71558) and phospho-mTOR(Cat#ab109268) were from Abcam. C225 (Cat #MABF120), was from EMD Millipore for EGFR immunoprecipitation studies. LC3 (Cat#NB100-2220) was from Novus biologicals. EGFR TKI AEE788 (Cat# S1486), Akt inhibitor, MK2206 (Cat#S1078), were obtained from Selleckchem. Plasmid-based transfections and siRNA-based transfections were performed using Lipofectamine 3000 (Invitrogen) and Lipofectamine RNAiMax (Invitrogen), respectively. Protein A/G beads (Santa Cruz Biotechnology) were used for immunoprecipitation.

Total cell lysates (20 μg) were separated using sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis (PAGE) and were transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). Membranes were blocked in 5% non-fat milk and incubated with primary antibodies overnight at 4°C. Membranes were then washed and incubated with horseradish peroxidase-conjugated secondary antibodies (anti-mouse or anti-rabbit) at room temperature for 2 h. Proteins were visualized using SuperSignal West Pico Luminol/Enhancer solution (Thermo Fisher Scientific Inc., Rockford, IL, USA). Akt1,2,3 primary antibody (Cell Signaling, Danvers, MA, USA) was diluted 1:200 in 0.1% milk/Tris-buffered saline and Tween-20 (TBS-T). β-actin (Bioworld Technology Inc., St. Louis Park, MN, USA), mTOR, and phospho-mTOR (Abcam, Cambridge, MA, USA) primary antibodies were all diluted 1:500 in TBS-T. Anti-mouse or anti-rabbit secondary antibodies (1 mg/mL, Dako North America Inc., Carpinteria, CA, USA) were diluted 1:1,000 in 5% milk/TBS-T.

Western blot was performed according to a previously described protocol [14 ]. The ischemic brain tissues were homogenized and centrifuged then the supernatants were collected. Total protein content was determined by BCA assay (Beyotime, China) according to the manufacturers’ instructions. 20 µg protein of supernatant aliquots were run on SDS-PAGE (10%). Bovine serum albumin (3%) was blocked in 0.2% to 0.4% TBST for 60 min and incubated in the primary antibodies in 0.5% BSA in 0.1% TBST O/N at 4 °C. Primary antibodies used in this experiment were rabbit monoclonal antibodies following: LC3 (Abcam, USA), p62 (Cell Signaling Technology, USA), Atg7 (Beyotime, China), PI3K (1:1,000, Cell Signaling Technology, USA), mTOR (Abcam, USA), Phospho-mTOR (Abcam, USA), Akt (Cell Signaling Technology, USA), Phospho-Akt (Cell Signaling Technology, USA), and GAPDH (Abcam, USA). Next, a goat anti-mouse IgM (1:5,000) or antirabbit IgG (1:5,000) were incubated with the membranes for 1 h. The bands were semi-quantified by densitometry using ImageJ software.

Formalin‐fixed, paraffin‐embedded (FFPE) tissue sections were deparaffinized and rehydrated by using xylene and ethanol, respectively. After retrieval with citrate buffer at pH 6.0, the endogenous peroxidase activity of specimens was quenched by peroxidase‐blocking solution (Dako). The sections were incubated with the primary antibody against the target protein as indicated below: MYC (Abcam, ab32072), CEBPD (Abcam, ab184911), HK2 (Cell Signaling, 2867), FBXW7 (Abcam, ab105752), phospho‐AKT (Cell Signaling, 4060), phospho‐mTOR (Abcam, ab51044), phospho‐4E‐BP1 (Cell Signaling, 9644), phospho‐RPS6 (Abcam, ab80158), and MKI67 (Abcam, ab66155), followed by the incubation of a secondary antibody (REAL EnVision/HRP, rabbit/mouse [ENV], Dako). Staining was visualized with EAL DAB+ Chromogen diluted in REAL Substrate Buffer (Dako). Hematoxylin was used for the nuclear stain. Finally, the slices were dehydrated by soaking in various concentrations of ethanol and were mounted in UltraCruz Aqueous Mounting Medium with DAPI. The IHC staining results were examined with an optical microscope and quantified into H‐scores by three expert pathologists (Chien‐Feng Li, Tzu‐Ju Chen and Wan‐Shan Li ) as previously mentioned.²² Detailed information is shown in Supporting Information.

Immunohistochemical staining was performed using the SABC kit (Maxim, Fuzhou, China) according to the manufacturer’s instructions. Briefly, the pancreas tissue sections were incubated in primary antibodies for CK19 (Abcam, Cambridge, MA, USA), phospho-STAT3 (CST, Danvers, MA, USA), phospho-AMPK (CST, Danvers, MA, USA), phospho-mTOR (Abcam, Cambridge, MA, USA), and α-SMA (Abcam, Cambridge, MA, USA) overnight at 4 °C; then, sections were incubated in the appropriate biotinylated secondary antibody for 30 min at room temperature, followed by 30 min of incubation with streptavidin peroxidase (Dako LSAB + HRP kit). After rinsing, the results were visualized using DAB, and the slides were counterstained with hematoxylin.

Cell lysates were extracted from cells using RIPA lysis buffer. Proteins were separated by SDS–PAGE (10% polyacrylamide), transferred to a PVDF membrane (Millipore), and blocked in 5% dry milk in Tris-buffered saline with Tween 20 (TBST; Applygen Technologies, Beijing, China). Membranes were incubated with primary antibody, and target proteins were detected with enhanced chemiluminescence (ECL) using ECL detection reagent (Applygen Technologies, Beijing, China). Primary antibodies included the following: AKT (Cell Signaling Technology Cat# 9272), phospho-AKT (Cell Signaling Technology Cat# 4060), AMPKα (D5A2) (Cell Signaling Technology Cat# 5831), phospho-AMPKα (Thr172) (40H9) (Cell Signaling Technology Cat# 2535), mTOR (Cell Signaling Technology Cat# 2983), phospho-mTOR (Abcam Cat# ab109268), p70S6K (Proteintech Cat# 14485-1-AP), phospho-p70S6K (Cell Signaling Technology Cat# 9234), 4eBP1 (Proteintech Cat# 60246-1-Ig), phospho-4eBP1 (Cell Signaling Technology Cat# 2855), GAPDH (Proteintech Cat# 60004-1-Ig), CDK1 (Santa Cruz Biotechnology Cat# sc53219), CDK4 (Cell Signaling Technology Cat# 12790), CDK6 (Abcam Cat# ab124821), Rb (Cell Signaling Technology Cat# 9309), and phospho-Rb (Ser795) (Cell Signaling Technology Cat# 9301).

To collect whole-cell lysates, cells were lysed in RIPA lysis buffer supplemented with a Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA), and the cell lysates were centrifuged at 12,000 rpm. The supernatant was collected, mixed with loading buffer, and boiled for 15 min to disengage the protein secondary structure. The mixture was resolved on SDS-PAGE gels (BioSci™ NewFlash Protein AnyKD PAGE; DAKEWE, Beijing, China) and then transferred onto nitrocellulose membranes (GE life, Pittsburgh, PA, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies overnight at 4 °C. The bands were probed with the appropriate secondary antibodies and detected by enhanced chemiluminescence. The following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): PD-1 (D4W2J, 1:1000), phospho-AKT (Ser473) (D9E, 1:2000), phospho-mTOR (Ser2448) (D9C2, 1:1000), phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E, 1:2000), AKT (pan) (C67E7, 1:1000), mTOR (7C10, 1:1000), S6 Ribosomal Protein (54D2, 1:1000), β-TrCP (D13F10, 1:1000), and β-Actin (8H10D10, 1:1000), except anti-PD-L1 antibody [EPR19759] (ab213524; Abcam, UK, 1:1000).