Phospho ulk1 s757

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Phospho-ULK1 S757 is a primary antibody that specifically recognizes ULK1 protein phosphorylated at serine 757. ULK1 is a serine/threonine protein kinase that plays a crucial role in the initiation of autophagy.

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Phospho-ULK1 (18 citations)

8 protocols using phospho ulk1 s757

Comprehensive Signaling Pathways Analysis

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Antibodies to BAX, BAK, BCL-XL, CHOP, c-FLIP, IRE1, RIP1, iNOS, FADD, Cathepisin B, mTOR, phospho-mTOR S2448 and S2481, phospho-RAPTOR S792, TSC2 T1426, PTEN, phospho-PTEN S380, ATF6, eNOS, AIF, XBP1, NOXA, PUMA, ATG5 phospho-ATG13 S318, Beclin-1, AKT, phospho-AKT T308, eiF2α, phospho-eiF2α S51, phospho p65 S536, ATF4, PGKI and II, TRX, SOD, ATM, phospho-ATM S1981, AMPKα, phospho-AMPK T172, phospho-ULK1 S757, S317, STAT3, STAT5, p70 S6K, phospho-ERK1/2, GRP78, HSP70 and HSP90, phospho-γH2AX, were purchased from Cell Signaling Technology, (Danvers, MA). PERK, CD95 and caspase 9 antibodies, were purchased from Santa Cruz Biotechnology, (Dallas, TX).

Roberts J.L., Poklepovic A, & Booth L. (2017). Curcumin interacts with sildenafil to kill GI tumor cells via endoplasmic reticulum stress and reactive oxygen/ nitrogen species. Oncotarget, 8(59), 99451-99469.

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Western Blot Analysis of mTOR Signaling

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Myocardial lysates were derived from flash frozen tissue (Cell Signaling Technology #9803) and protein concentration determined by BCA (Pierce). Samples were prepared in SDS Tris-Glycine buffer (Life Technologies) and run on TGX 7.5% or 4–15% Tris-Glycine Gels (Bio-Rad) and blotted onto a nitrocellulose membrane. The following primary antibodies were used in this study: phospho-p70 S6K (T389) #9205 lot 26 used at 1:1,000, p70 S6K #9202 lot 20 used at 1:1,000, phospho-4EBP1 (S65) #9451 lot 14 used at 1:1,000, 4EBP1 #9452 lot 12 used at 1:1,000, phospho-Ulk-1 (S757) #1420 clone D706U lot 4 used at 1:500, Ulk-1 #8054 clone D8H5 lot 5 used at 1:500, TSC2 #3612 clone D93F12 lot 6 used at 1:1,000 (Cell Signaling Technology), phospho-TSC2 (S1365) #120718 lot NFSA12072OAH used at 1:500 (NovoPro Labs), and P62 #ab109012 lot GR12843–70 used at 1:1,000 (Abcam), and a total protein stain #926–11016 lot C80522–02 used at 5 ml/membrane (Li-Cor). Antibody binding was visualized by infrared imaging (Odyssey, Li-Cor) and quantified with Li-Cor Image Studio Software 3.1.

Oeing C.U., Jun S., Mishra S., Dunkerly-Eyring B.L., Chen A., Grajeda M.I., Tahir U.A., Gerszten R.E., Paolocci N., Ranek M.J, & Kass D.A. (2021). MTORC1-Regulated Metabolism Controlled by TSC2 Limits Cardiac Reperfusion Injury. Circulation research, 128(5), 639-651.

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Antibody Validation for Autophagy Signaling

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Phospho-AMPKα T172 (2531; 1:1000), AMPKα1 (2532; 1:1000), phospho-ULK1 S757 (14202; 1:1000), phospho-beclin 1 S15 (13825; 1:1000), beclin 1 (3495; 1:1000), PKCα (2056; 1:1000), ULK1 (8054; 1:1000), ubiquitin (3933; 1:1000), 4E-BP1 (9452; 1:1000), phospho-4E-BP1 Thr37/46 (2855; 1:1000), mTOR (2983; 1:1000), phospho-mTOR S2448 (5536; 1:1000), phospho-(Ser/Thr) PKC substrate (6967; 1:1000), β-tubulin (2146; 1:3000), HA (2999; 1:3000), GFP (2956; 1:2000), and Myc (2278; 1:2000) antibodies were from Cell Signaling Technology, Danvers, MA. LC3 antibody (NB100-2220; 1:7000) was from NOVUS Biologicals, Littleton, CO. p-Atg13 S318 (600-401-C49S; 1:1000) was from Rockland Immunochemicals, Limerick, PA. β-Actin antibodies (sc-47778; 1:10000) were from Santa Cruz Biotechnology, Dallas, Texas. P62 (SQSTM1) antibody (ab56416; 1:4000), SNAP29 (ab56566; 1:1000), LAMP2a (ab18528; 1:3000), and VAMP8 (EP2629Y; 1:1000) were from Abcam, Cambridge, MA. GST (E022040-01; 1:10000) was from EARTHOX Life Sciences, Millbrae, CA, and Flag (SLBJ7864V; 1:2000) and STX17 (HPA001204; 1:1000) were purchased from SIGMA-Aldrich, St. Louis, MO. FIP200 (17250-1-AP; 1:1000) was from Proteintech, Rosemont, IL. Total p-S, T (612549; 1:4000) antibody was from BD Biosciences, San Jose, CA. Anti-phospho ULK1 S423 antibody (1:100) was generated and affinity purified by ABclonal Technology, Wuhan, China.

Wang C., Wang H., Zhang D., Luo W., Liu R., Xu D., Diao L., Liao L, & Liu Z. (2018). Phosphorylation of ULK1 affects autophagosome fusion and links chaperone-mediated autophagy to macroautophagy. Nature Communications, 9, 3492.

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Cell Lysis and Immunoblotting Protocol

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Cells were lysed using lysis buffer containing 20 mM Tris pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 50 mM NaF, 1 mM DTT, with protease inhibitor cocktail (Sigma #P8340), phosphatase inhibitor cocktail #2 (Sigma #P5726), and #3 (Sigma #P0044) used at 1:100 each. Western blots were performed using the following antibodies purchased from Cell Signaling Technology and used at 1:1000 dilution unless otherwise indicated: phospho-p70 S6 Kinase T389 (CST #9234 1:2000), p70 S6 Kinase (CST #2708, 1:2500), phospho-4E-BP1 T37/46 (CST #2855), 4E-BP1 (CST #9644, 1:5000), phospho-ULK1 S757 (CST #14202), ULK1 (CST #8054), phospho-S6 S235/236 (CST #4858 1:5000), phospho-S6 S240/244 (CST #5364 1:5000), S6 (CST #2317), Cyclin E1 (CST #20808), Cyclin A2 (CST #4656), Cyclin B1 (CST #4138), Cyclin D2 (CST #3741), phospho-TSC2 T1462 (CST #3617), phospho-GSK3 S9/21 (CST #9331), TSC2 (CST #4308), GAPDH (CST #5174, 1:5000), phospho-Akt T308 (CST #13038), phospho-Akt S473 (CST #4060 1:5000), Akt (CST #4691), phospho-Erk Y202/204 (CST #9106), Erk (CST #9102), phospho-Wee1 S642 (CST #4910), Wee1 (CST #13084), phospho-CDK1 Y15 (CST #4539), CDK1 (CST #9116), phospho-Chk1 S345 (CST #2348), phospho-Chk1 S296 (CST #90178), Chk1 (CST #2360), LC3 A/B (CST #12741). Immunoblots were quantified using ImageJ^{47 (link)} and normalized to the first lane in each respective blot.

Joshi J.N., Lerner A.D., Scallo F., Grumet A.N., Matteson P., Millonig J.H, & Valvezan A.J. (2024). mTORC1 activity oscillates throughout the cell cycle promoting mitotic entry and differentially influencing autophagy induction. bioRxiv.

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Immunoblotting of GFP-WIPI1 U-2 OS Cells

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GFP-WIPI1 expressing U-2 OS cells were treated as described above for WIPI1 puncta formation analysis, except that after the 3 h treatment period, cells were lysed with hot 2x Laemmli buffer (50 mM Tris pH 6.8, 1.25 mM EDTA pH 8.0, 12.5% glycerine, 2% SDS, 50 mM DTT, 2.5% β-mercaptoethanol, 0.025% bromophenol blue), scraped into Eppendorf tubes, and sheered with a 23G injection needle. 40 μl of the extracts was loaded on a 10% or 15% SDS-PAGE gel, blotted on to a PVDF membrane (Millipore, IPVH00010), and incubated with the following antibodies: SGK1 (Cell Signaling, 12103), LC3 (NanoTools, 0231–100/LC3-5F10), phospho-ULK1 (S757) (Cell Signaling, 6888), ULK1 (Cell Signaling, 8054), α-tubulin (Sigma-Alderich, T6074), anti-mouse IgG-HRP (Cell signaling, 7076), and anti-rabbit IgG-HRP (Cell Signaling, 7074). Subsequently, ECL detection was performed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, 34096). Images were taken using the Fusion SL Vilber Lourmat device.

Zuleger T., Heinzelbecker J., Takacs Z., Hunter C., Voelkl J., Lang F, & Proikas-Cezanne T. (2018). SGK1 Inhibits Autophagy in Murine Muscle Tissue. Oxidative Medicine and Cellular Longevity, 2018, 4043726.

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Antibody-based Analysis of Signaling Pathways

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Antibodies to Akt, phospho-Akt(S473), phospho-Akt(T308), phospho-Tuberin/TSC2(T1462), Tuberin/TSC2, GSK-3β, phospho-GSK-3β(S9), mTOR, Raptor, phospho-4E-BP1(T37/46), p70S6K, phospho-p70S6K(T389), LC3A/B, ULK1, phospho-ULK1(S757) and Anti-Rabbit IgG Fab2 Alexa Fluor (R) 488 conjugate were obtained from Cell Signaling Technology (Beverly, MA, USA). β-Actin and HA-probe antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Piperlongumine was obtained from Indofine Chemical Company (Hillsborough, NJ, USA). Chloroquine diphosphate, Puromycin dihydrochloride, Hexadimethrine bromide and N-Acetyl-L-Cysteine were obtained from Sigma-Aldrich (St Louis, MO, USA). Bafilomycine A1 was obtained from LC Laboratories (Woburn, MA, USA).

Makhov P., Golovine K., Teper E., Kutikov A., Mehrazin R., Corcoran A., Tulin A., Uzzo R.G, & Kolenko V.M. (2014). Piperlongumine promotes autophagy via inhibition of Akt/mTOR signalling and mediates cancer cell death. British Journal of Cancer, 110(4), 899-907.

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Western Blot Analysis of Cellular Signaling

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Cells were lysed in buffer containing 50 mM Tris pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol, 1 mM sodium orthovanadate, 20 mM sodium fluoride, 10 mM beta-glycerophosphate, 10 mM sodium pyrophosphate, 2 ug ml⁻¹ aprotinin, 2 ug ml⁻¹ leupeptin and 0.7 ug ml⁻¹ pepstatin. Western blot analysis was performed using standard protocols, and the following commercial antibodies were used as probes: ASNS (Proteintech 14681-1-AP, 1:1000), ATF4 (Cell Signaling 11815, 1:500), phospho-T389 S6 kinase (Cell Signaling 9234, 1:500), S6 kinase (Cell Signaling 2708, 1:1000), phospho-S235/235 S6 ribosomal protein (Cell Signaling 4858, 1:3000), S6 ribosomal protein (Cell Signaling 2217, 1:1000), phospho-S1859 CAD (Cell Signaling 70307, 1:500), CAD (Cell Signaling 11933, 1:1000), 4E-BP1 (Cell Signaling 9644, 1:500), phospho-S757 ULK1 (Cell Signaling 14202, 1:1000), ULK1 (Cell Signaling 8054, 1:1000), phospho-T172 AMPKα (Cell Signaling 2535, 1:500), AMPKα (Cell Signaling 2532, 1:1000), PHGDH (Cell Signaling 13428, 1:500), β-Actin (Cell Signaling 4970, 1:1000), PSAT1 (Novus 89-004-606, 1:500), and a-tubulin (Sigma T6074, 1:10,000).

Krall A.S., Mullen P.J., Surjono F., Momcilovic M., Schmid E.W., Halbrook C.J., Thambundit A., Mittelman S.D., Lyssiotis C.A., Shackelford D.B., Knott S.R, & Christofk H.R. (2021). Asparagine couples mitochondrial respiration to ATF4 activity and tumor growth. Cell metabolism, 33(5), 1013-1026.e6.

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Antibody-based Western Blot Analysis

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Antibodies used for Western blot were purchased from Cell signaling technology, Danvers, MA, USA: AKT (pan) (C67E7), phospho-S473-AKT (D9E), phosphor-T308 AKT (244F9), AMPKα, phospho-T172-AMPKα (40H9), p70-S6K, phospho-T389-p70-S6K, S6P (5G10), phospho-S235/236-S6P, Raptor (24C12), Rictor (53A2), elF2α, phospho-S51-elF2α (119A11), ULK1 (D8H5), phospho-S757-ULK1, phospho-S555-ULK1 (D1H4), 4-EBP1 and LC3B (D11) XP. Antibodies used for immunocytochemistry: elF3η (C-5) was purchased from Santa Cruz Biotechnology, Dallas, TX, USA, and G3BP1 was purchased from Thermo Fischer Scientific, Waltham, MA, USA.

Hussain I., Sureshkumar H.K., Bauer M, & Rubio I. (2023). Starvation Protects Hepatocytes from Inflammatory Damage through Paradoxical mTORC1 Signaling. Cells, 12(12), 1668.

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