Anti p mtor

Flow cytometry: commercial antibodies anti-CD86-FITC (clone 2231 FUN-1), CD274 (PD-L1)-FITC (clone MIH1), CD273 (PD-L2)-PE (clone MIH-18), HLA-DR-PE-Cy7 (clone L243), IFN-γ-FITC (clone 4SB3) were purchased from BD Biosciences; CD83-PerCP-Cy5.5 (clone HB15a) was purchased from Beckman Coulter; CD80-FITC (clone MAB104), CD40-PerCP-eFluor710 (clone 5C3), CD1a-PE-Cy7 (clone HI149) and CD4-PE-Cy7 (clone RPA-T4) were purchased from eBioscience; TLR2-FITC (clone T2.5), TIM-3-PE (clone F38-2E2), IL-10-PE (clone JES3-9D7), KI-67-PE (clone Ki-67) were purchased from BioLegend; CD14-PE-DL594 (clone MEM-15), CD11c-APC (clone BU15), CD3-AF700 (clone MEM-57), CD8-PE-Dy590 (clone MEM-31) were purchased from Exbio; CD85k (ILT-3)-PE (clone 293623), CD85d (IL-T4)-FITC (clone 287219) were purchased from R&D Systems. For western blot, anti-p-p38 MAPK, anti-p-ERK1/2, anti-p-JNK/SAPK, anti-p-IκB-α, anti-IDO, anti-p-mTOR, anti-p-STAT3, anti-p-p70S6K, anti-p38 MAPK, anti-ERK1/2, anti-JNK/SAPK and anti-STAT3 Ab were purchased from Cell Signaling Technology; anti-actin was from BioLegend.

The whole cells were washed and lysed in RIPA buffer (89900, Thermo Scientific) supplemented with PMSF (phenylmethylsulfonyl fluoride) and protease inhibitors. Protein concentrations were determined with a BCA assay kit (Pierce, Rockford, IL, USA). Then the lysates were mixed with loading buffer, analyzed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocked with 5% skim milk in TBST at room temperature for 1 h, the membranes were incubated with different primary antibodies, including anti-DDX54 (1:1000, Proteintech, China), anti-P65, anti-p-P65, anti-AKT, anti-p-AKT, anti-mTOR, anti-p-mTOR, (1:1000, Cell Signaling Technology, Danvers, MA, USA), and anti-GAPDH (1:5000, Yeason, China) in 5% milk/TBST buffer at 4°C overnight, and then probed with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:5000, Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 1 h. After washing three times with TBST, the membrane was developed with enhanced chemiluminescent plus substrate (Merck Millipore, Billerica, MA, USA), and the signal was recorded by Fluorchem E System (ProteinSimple, Santa Clara, CA, USA).

Liver homogenates and cells were lysed in ice-cold RIPA buffer (50 mM Tris HCl (pH 7.4), 250 mM NaCl, 1% Nonidet P-40, and protease inhibitor cocktail). The protein samples (50 µg) were separated by 4%–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto PVDF membranes. After blocking with 5% BSA or 5% non-fat dry milk, the membranes were incubated overnight with the following primary antibodies: anti-p-mTOR, anti-mTOR, anti-p-p70s6kinase, anti-p70s6kinase, anti-p-4EBP-1, anti-4EBP-1, anti-CHOP, anti-p-eIF2α, and anti-eIF2α (Cell Signaling Technology, Boston, MA, USA), anti-ubiquitin, anti-GRP78, anti-p-PERK, anti-PERK, anti-Bax, anti-lamp-1, anti-Tom20, anti-cathepsin B, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p62 (SQSTM1; MBL International, Woburn, MA, USA), and anti-LC3II (Novus Biologicals, Littleton, CO, USA). Immunoreactive bands were visualized using the ECL Western blotting protocol (Bio-Rad). Finally, the membranes were exposed to imaging film (Kodak BioFlexEcono Scientific Supplies, Citrus Heights, CA, USA), after which the film was developed using a Kodak X-OMAT 1000A Processor. Densitometric analysis of the bands was conducted using the Image J software (NIH, Bethesda, MD, USA).

Protein was extracted from MGC-803 cells at different groups and tumor tissues using RIPA buffer containing PMSF. Proteins (50 µg) were resolved on 10% SDS-PAGE and electrotransferred on polyvinylidene fluoride membranes (Invitrogen, CA, USA). The primary antibodies used here were anti-PI3K, anti-p-PI3K, anti-AKT, anti-p-AKT, anti-mTOR, anti-p-mTOR, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-p53, anti-CDK4, anti-CyclinD1 or anti-beta-actin from Cell Signaling Technology or Abcam. Then, the membranes were incubated with secondary antibody and target proteins were analyzed by ImageLab software (Bio-Rad, CA, USA) [25 (link)].

PDI-Flag, PHB2-Flag plasmids constructed with pcDNA3.1(+) cloning vector were purchased from GENEWIZ company (Suzhou, China). LC3-His, PDI-Myc, plasmids constructed with pET-28a (+) cloning vector was purchased from Genomeditech Company (Shanghai, China). The antibodies were purchased from Proteintech (IL, USA) as follows: anti-PDI (11245-1-AP), anti-PEPK (20582-1-AP), anti-ATF6 (24169-1-AP), anti-TOM20 (11802-1-AP), anti-TOM40 (18409-1-AP), anti-Calnexin (10427-1-AP), anti-IRE1 (27528-1-AP), anti-PARP (13371-1-AP), anti-AKT (60203-2-Ig), anti-Actin (66009-1-Ig), anti-LC3 (14600-1-AP). The antibodies were purchased from Cell Signaling (MA, USA) as follows: anti-P62 (#39749), anti-PHB2 (#14085), anti-PINK1 (#6946), anti-mTOR (#2972), anti-p-mTOR (#5536), anti-Bcl-2 (#4223), anti-Caspase-7 (#9492), anti-p-AKT (#4060), anti-LC3A/B (#4108). The antibodies were purchased from Santa (CA, USA) as follows: anti-GRP78 (sc-13539), and anti-PHB2 (sc-133094). The antibodies were purchased from Beyotime (Shanghai, China) as follows: anti-FLAG (AF0036), anti-HIS (AF5060), and anti-MYC (AF0033).

Fresh tissues and cells were lysed with cell lysis buffer (Beyotime Biotechnology) and western blot was performed as described previously [18 (link)]. Briefly, 40 μg total proteins were applied to separation with SDS–PAGE gel. After the electrophoresis, the proteins were transferred to PVDF membranes (Millipore), followed by blocking in the TBST buffer containing 5% fat-free milk. The membranes were then incubated with indicated antibodies overnight at 4 °C, and then washed and incubated with HRP-conjugated secondary antibodies (Zhongsanjinqiao) for 2 h at room temperature and finally visualized using Chemiluminescent ECL reagent (Vigorous Biotechnology). The following antibodies were used in this work: Anti-GAPDH (Cell Signaling Technology), anti-JMJD2A (Cell Signaling Technology), anti-Histone H3 (Santa Cruz Biotechnology), anti-H3K9me3 (Abcam), anti-H3K36me3 (Abcam), anti-mTOR (Cell Signaling Technology), anti-p-mTOR (Cell Signaling Technology), anti-Akt (Cell Signaling Technology), anti-p-Akt Thr308 (Cell Signaling Technology), anti-S6K1 (Cell Signaling Technology), anti-p-S6K1 (Cell Signaling Technology).