Anti mtor

In order to detect the protein expressions of mTOR, phospholipase C gamma 1 (PLCG1) and SHC adaptor protein 1 (SHC1), total proteins were extracted using lysis buffer, separated on 12% SDS-PAGE gels and blotted on cellulose membranes. The membranes were immunoblotted with a secondary antibody at room temperature for 1 hour after hybridization with a monoclonal antibody at 4 °C overnight. Finally, enhanced chemiluminescence (ECL kit, Santa Cruz Biotechnology) was used for visualization and a Quantity One system (Bio-Rad, Hercules, CA, USA) was applied for analysis. Primary antibodies included anti-mTOR (dilution 1:200, Santa Cruz Biotechnology), anti-PLCG1 (dilution 1:1000, ThermoFisher Scientific), anti-SHC1 (dilution 1:2000, Abnova) and anti-GAPDH (dilution 1:1000, Santa Cruz Biotechnology).

Western blotting was performed as previously described 21 (link).
The antibodies used were as follows: anti-Msi1 (1:1000 dilution, Abnova, Taipei, Taiwan), anti-PTEN (1:1000 dilution, Santa Cruz, CA, USA), anti-PI3K (1:1000 dilution, Santa Cruz, CA, USA), anti-p-AKT (1:1000 dilution, Cell Signaling Technology), anti-AKT1 (1:1000 dilution; Santa Cruz, CA, USA), anti-mTOR (1:1000 dilution, Santa Cruz, CA, USA), anti-BAK (1:500 dilution, Santa Cruz Biotechnology), anti-Bcl-2(1:1000 dilution, Santa Cruz, CA, USA), anti-GAPDH (1:1000 dilution, Santa Cruz, CA, USA), and the secondary antibody coupled to horseradish peroxidase (Goat aiti-Mouse:1:10000 dilution; Goat anti-Rabbit: 1:15000, Thermo Fisher Scientific Inc., New York, NY, USA). The proteins were visualized with an enhanced chemiluminescence reagents (Millipore, Billerica, MA, USA) with the protein imprinting imaging system (Tanon 5200, China). GAPDH was used as the control and for quantification.

(RS)-3,5-DHPG and dynasore were purchased from Tocris Bioscience (Bristol, UK). AP5 (D-2-amino-5-phosphonopentanoate), MPEP, cholesterol, and methyl-β-cyclodextrin (Mβ-CD) were obtained from Sigma (St. Louis, MO, USA). We used the following antibodies: anti-FMRP (MAB2160; Millipore, Billerica, MA, USA), anti-Cav1 (ab2910, Abcam, Cambridge, UK), anti-GluA1 (ab31232; Abcam), anti-GluA2 (ab20673; Abcam), anti-pan-cadherin (C1821; Sigma), anti-ERK1/2 (sc514302; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pERK1/2 (sc81492; Santa Cruz Biotechnology), anti-mTOR (sc517464; Santa Cruz Biotechnology), anti-pmTOR (sc293133; Santa Cruz Biotechnology), and anti-β-actin (A5316; Sigma).

Icariin, L-asparagine (Asn), 3-methyladenine (3-MA), Oil Red O, MTT assay kit, uranyl acetate/lead citrate, and MDC were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, adipocyte differentiation medium, and gentamycin were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). The Annexin V-FITC apoptosis detection kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Anti-mTOR, anti-p-mTOR, anti-beclin-1, anti-AMPK, anti-p-AMPK, anti-p62, anti-LC3, and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Human ovarian cancer cell lines (SKOV-3, 2774 and OVCAR-3) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Human IFN-α and IFN-γ were obtained from Sigma (St. Louis, MO). The chemical doxazosin was also purchased from Sigma. The JAK1/2 kinase inhibitor INCB18424 (Ruxolitinib) and STAT1 inhibitor (NSC118218) were obtained from Selleck Chemicals (Houston, TX), and stock solutions were prepared in DMSO. NSC74859 (S31-201), a specific STAT3 inhibitor, was purchased from Calbiochem Chemicals (La Jolla, CA). The following primary antibodies were used in this study: anti-JAK1, anti-phospho-JAK1, anti-JAK2, anti-phospho-JAK2, anti-STAT1, anti-phospho-STAT1, anti-STAT3, anti-phospho-STAT3, anti-caspase-3, anti-cMyc, anti-Bcl-2, anti-Bax, anti-p53, anti-survivin, and anti-COX-2 (Cell Signaling, Beverly, MA), anti-PARP, anti-XIAP (BD Biosciences, San Jose, CA), anti-cyclin D1, anti-CDK4, anti-Akt, anti-phospho-Akt, anti-TYK2, anti-phospho-TYK2, anti-PI3K, anti-phospho-PI3K, anti-mTOR, anti-phospho-mTOR, anti-PKCδ, anti-phospho-PKCδ, anti-STAT2, anti-phospho-STAT2, anti-p70S6K, and anti-phospho-p70S6K (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p21, and anti-p27 (Oncogene, San Diego, CA).

The samples were lysed in RIPA buffer to obtain soluble proteins. The precipitates were further cleaved with RIPA buffer containing SDS and urea to obtain insoluble proteins. The protein concentration was determined via bicinchoninic acid assay. The proteins were then isolated and transferred to polyvinylidene fluoride membranes. Next, the membranes were incubated with primary antibodies, including anti-Aβ1–16 (BioLegend, SIG-39300), anti-TFEB (Proteintech, 13372-1-AP), anti-phospho-MAPK (Cell Signaling Technology, 4370), MAPK (Cell Signaling Technology, 4695), anti-p-Akt (Cell Signaling Technology, 4060 T), anti-Akt (Cell Signaling Technology, 4685 s), anti-ribosomal protein S6 (RPS6; Santa Cruz Biotechnology, sc-74459), anti-p-RPS6 (Cell Signaling Technology, 2211), anti-mTOR (Santa Cruz Biotechnology, sc-517464), anti-p-mTOR (Santa Cruz Biotechnology, sc-293133), anti-p62 (Proteintech, 18420-1-AP), anti-LC3A/B (Cell Signaling Technology, 12741S), anti-LAMP1 ([Santa Cruz Biotechnology, sc-20011] or [Abcam, ab24170]), and anti-β-actin (Sigma-Aldrich, A5316). The binding of the primary antibodies was visualized using HRP-conjugated secondary antibodies. Furthermore, grayscale analysis was conducted using FIJI ImageJ (National Institutes of Health).