Vps34

The following antibodies were used: Tsc2, Atg5, Vps34, pS6 ribosomal, S6 ribosomal protein, pAkt, total Akt (Cell Signaling Technology), LC3 (Novus Biologicals), actin (Sigma-Aldrich). Rapamycin and Torin1 were purchased from LC Laboratories. EIPA was purchased from Sigma-Aldrich. SAR405 was purchased from ApexBio (Houston, TX). shRNA targeting Vps34 were obtained from Sigma-Aldrich: TRCN0000322313 (shPI3KC3-1) and TRCN0000350673 (shPI3KC3-2). Pools of cells with stable shRNA expression were selected using puromycin (10ug/ml) and knockdown of Pik3c3 was validated using qRT-PCR and immunoblotting.

The DCs were lysed in a RIPA buffer (Cell Signaling Technologies, Danvers, MA, USA) in the presence of a cocktail of protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Equal amounts of total proteins were loaded onto SDS-PAGE gel and blotted onto a nitrocellulose membrane (BIO-RAD, Hercules, CA, USA). The membranes were incubated overnight, at 4 °C, with the following primary antibodies: HIF-1α (BD Biosciences, San Jose, CA, USA); phmTOR, mTOR, phULK1 ser 757, SQSTM1/p62, phAKT and AKT, Mcl-1, Vps34, Beclin-1, Atg5, phERK, α-Tubulin (Cell Signaling Technologies, Danvers, MA, USA); ULK1 and LAMP1 (Santa Crutz Biotechnology, Dallas, TX, USA) and β-actin (Sigma-Aldrich). Anti-mouse IgG HRP and anti-rabbit IgG-HRP (Cell Signaling Technologies, Danvers, MA, USA) were used as secondary antibodies. Images were acquired by ChemiDoc™ MP System and blot quantification was performed by using Image Lab software (BIO-RAD, Hercules, CA, USA).

Protein lysates were harvested from macrophages using lysis buffer containing 50 mM Tris–Cl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA-free protease inhibitor cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride (PMSF). 50 μg of lysates were separated by SDS-PAGE and transferred to PVDF membrane (Millipore) for western blot analysis with specific primary antibodies (1:1000) for Ubiquitin (abcam, cat#ab13493), Msl2(CST,cat#ab44006), Outlin (abcam, cat# ab211328), p62 (Cell Signaling Technology, cat#ab16177), PI3Kp110α (Cell Signaling Technology,cat#ab4255), PI3K-p110β(Cell Signaling Technology,cat#ab3011), PI3Kp110δ(Cell Signaling Technology,cat#ab5405), Vps34(Cell Signaling Technology,cat#ab4263, AKT(Cell Signaling Technology,cat#ab4685), p-AKT(Ser473) (Cell Signaling Technology,cat#ab4060), mTOR(Cell Signaling Technology,cat#ab2972), Phospho-mTOR (Ser2448) (Cell Signaling Technology,cat#ab5563) and LC3 (Cell Signaling Technology, cat#ab4108), QKI (Sigma), LC3-II(Cell Signaling Technology, cat#ab2775), flag(Sangon, cat#D191041), HA(Sangon, cat#D191044), and Myc(Sangon, cat#D199941), Rnf6 (Thermo, cat#PA5-59044), EDC4 (proteintech, cat#D17737-1-AP) and β-actin(Sangon, cat#D191047). Immunolabelled proteins were detected by using appropriate HRP-conjugated secondary antibodies (Thermo, cat#31460), followed by visualization with ECL (Sangon).

General laboratory reagents were obtained from Thermo Fisher Scientific (Waltham, MA, USA) and Sigma‐Aldrich (Saint Louis, MO, USA). Antibodies against LC3B (#2775S), PARP (#9542S), cleaved caspase‐3 (#9661S), USP7 (#4833S), ubiquitin (#3933S), HA‐tag (#3724S), Vps15 (#14580S), and Vps34 (#3811S) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against NEK2 (sc‐55601), Beclin‐1 (sc‐48381), and phosphorylated mTOR (Ser 2448) (sc‐293133) were obtained from Santa Cruz Biotechnology, Inc (St Louis, MO, USA). β‐Actin antibody (66009‐1‐1g) and Flag antibody (20543‐1‐AP) were purchased from Proteintech Group (Rosemont, IL, USA). Phosphorylated Beclin‐1 (S90/93/96) antibody (#13232) and HRP‐conjugated secondary goat anti‐rabbit (#L3012) and goat anti‐mouse (#L3032) antibodies were purchased from Signalway Antibody (College Park, MD, USA). Alexa Fluor 488‐conjugated donkey anti‐rabbit second antibody (#A21202), Alexa Fluor 594‐conjugated donkey anti‐mouse second antibody (#A21207), and UltraPure™ LMP agarose were purchased from Thermo Fisher Scientific. IPKine HRP goat anti‐mouse IgG LCS (#A250112) was obtained from Abbkine Scientific Co (Wuhan, China).

Dulbecco's Modified Eagle Medium (DMEM) chemicals, monodansylcadaverine (MDC) and 7β-hydroxycholesterol were obtained from Sigma Aldrich Chemical (St. Louis, MO) as were all other reagents, unless specifically stated elsewhere. Fetal bovine serum (FBS) and penicillin-streptomycin were provided by Lonza (Basel, Switzerland). α-Asarone was purchased from Cayman chemical company (Ann Arbor, MI). Antibodies of eIF2α, phospho-eIF2α, Vps34, Atg5, Atg12-Atg5, Atg16L1, SQSTM1, NBR1, Atg7, Atg3, CHOP were obtained from Cell Signaling Technology (Danvers, MA). GADD34, beclin-1 antibodies were purchased from Abcam (Cambridge, UK). p150 antibody was obtained from Santa Cruz Biotechnology (Dallas, TX). LC3 antibody was purchased from MBL international corporation (Woburn, MA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and rabbit anti-mouse IgG were supplied from Jackson ImmunoResearch Laboratory (West Grove, PA).

NIH3T3 cells and COS-7 cells were purchased from American Type Culture Collection (ATCC) and used within six months after cells were ordered. These cell lines were not authenticated in our laboratory. Rapamycin, 5-Bromo-2′-deoxyuridine (BrdU) and cycloheximide were obtained from Sigma. PF-4708671 was purchased from Selleckchem. MG132 was purchased from CalBiochem. Antibodies directed against Myc-tag (9E10), Flag-tag, and HA-tag were obtained from Covance. Antibodies directed against VPS34 (cat#4263), VPS15 (cat#14580), Beclin 1 (cat#3495), pT389-S6K1 (cat#9234), S6K1 (cat#2708), pT37/46-4EBP1 (cat#2855), 4EBP1 (cat#9644), pS235/236-S6 (cat#4858), S6 (cat#2217), pErk1/2 (cat#4370), Erk1/2 (cat#4695), pS473-Akt(cat#4060), Akt (cat#4685), RheB (cat#13879), TSC1 (cat#6935), TSC2 (cat#4308), BrdU (cat#5292), and Cyclin E (cat#4129) were purchased from Cell Signaling Technology. Fluorescently tagged antibodies were obtained from Invitrogen. The class III PI3K lipid kinase ELISA kit was purchased from Echelon Biosciences. The RheB activation kit was purchased from Abcam. 4-6 weeks old SCID mice were ordered from National Cancer Institute (NCI), NIH (Bethesda, MD).