Anti raptor

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-Raptor is a primary antibody that recognizes Raptor, a component of the mammalian target of rapamycin complex 1 (mTORC1). Raptor is a regulatory-associated protein of mTOR and is critical for mTORC1 signaling.

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Raptor (86 citations) Rabbit anti-Raptor (6 citations) Anti-p-Raptor(Ser792) (4 citations) Anti-phospho-raptor (4 citations) Anti-Raptor (24C12), (4 citations) Anti-phospho-RAPTOR (Ser 792) (3 citations) Anti-Raptor antibody (2 citations)

30 protocols using anti raptor

Protein Expression Analysis by Immunoblotting

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Protein expression levels were evaluated by immunoblotting analysis as previously described [23 (link)]. Briefly, whole cell lysates (WCLs) were extracted using radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and protease/phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). The prepared WCLs were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The following primary antibodies were used: anti-cleaved caspase-3, anti-total caspase-3, anti-β-actin, anti-light chain 3 (LC3)-I/II, anti-p62, anti-Atg5, anti-Unc51 like autophagy activating kinase 1 (ULK1), anti-Beclin-1 (Abcam, Cambridge, MA, USA), anti-total mammalian target of rapamycin (mTOR), anti-phosphor-mTOR1 (p-2448S), anti-phosphor-mTOR2 (p2481S), antitotal-protein kinase B (Akt), anti-phosphor-Akt, anti-raptor, and anti-rictor (Cell Signaling Technology, Danvers, MA, USA).

Hong S.W., Lee J., Moon S.J., Kwon H., Park S.E., Rhee E.J, & Lee W.Y. (2024). Docosahexanoic Acid Attenuates Palmitate-Induced Apoptosis by Autophagy Upregulation via GPR120/mTOR Axis in Insulin-Secreting Cells. Endocrinology and Metabolism, 39(2), 353-363.

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Protein Modification and Regulation Assays

Cited 1 time

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NButGT, Ac-5SGlcNAc were provided by D. Vocadlo (Simon Fraser University)(48 (link)). SRT1720, EX-527, Compound C purchased from Selleck-Chemicals (Houston, TX). MG132 purchased from Sigma-Aldrich (St. Louis, MO). pcDNA3.1-SIRT1-Flag was gift from E. Verdin (Addgene plasmid#: 13812). pLenti4-HA-OGT (provided by K. Vosseller, Drexel University). Antibodies used: anti-actin, anti-FOXM1 and anti-RL2 from Santa Cruz Biotechnology; pERK1/2, anti-OGT and anti-O-GlcNAc from Sigma-Aldrich; anti-acetylated p53(K382), anti-AMPK, anti-pAMPK(S172), anti-ERK1/2, anti-MMP2, anti-pRaptor(792), anti-Raptor, anti-SIRT1 and anti-Ubiquitin(K48) from Cell Signaling (Danvers, MA); anti-Cdh1 and anti-p53 from Neobiolabs; anti-MMP9 from Novus-Biologicals (Littleton, CO); integrin α5 and α6 are from BD-Pharmingen (San Jose, CA).

Ferrer C.M., Lu T.Y., Bacigalupa Z.A., Katsetos C.D., Sinclair D.A, & Reginato M.J. (2016). O-GlcNAcylation regulates breast cancer metastasis via SIRT1 modulation of FOXM1 pathway. Oncogene, 36(4), 559-569.

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Western Blot Analysis of mTOR Pathway Proteins

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Lysates were prepared in 1x Dulbecco’s Phosphate Buffer Saline (DPBS—Invitrogen) containing 1% Nonidet P 40 substitue (NP40 –Signa-Aldrish, MO, USA) and protease inhibitor cocktail (Roche Diagnostics, IN, USA). Total protein was determined by Lowry’s method and 25 mg was loaded on a 4–12% gradient SDS–polyacrylamide gel electrophoresis (Invitrogen). Proteins were transferred to 2 μM nitrocellulose membrane using the Trans-blot turbot kit (Bio-Rad) and blotted over-night with anti-mTOR (rabbit polyclonal, abcam), anti-Rictor (rabbit polyclonal, Cell Signaling), anti-Raptor (rabbit polyclonal, Cell Signaling), anti-Atg5 (mouse monoclonal, Cell Signaling), anti-Atg7 (mouse monoclonal, Cell Signaling), anti-pS6K1 (rabbit polyclonal, Abcam), anti-S6K1 (rabbit polyclonal, Abcam), anti-peIF4E (Ser209) (rabbit polyclonal, Cell Signaling), anti-eIF4E (rabbit polyclonal, Cell Signaling), anti-E1/E2 (rabbit polyclonal, gift from Olivier Schwartz laboratory), anti-C (monoclonal antibody, gift from Olivier Schwartz laboratory), anti-GFP (rabbit polyclonal, Cell Signaling) or anti-GAPDH (rabbit polyclonal, Cell Signaling). Secondary HRP-coupled Abs was detected using ECL Plus (Amersham Pharmacia Biotech).

Joubert P.E., Stapleford K., Guivel-Benhassine F., Vignuzzi M., Schwartz O, & Albert M.L. (2015). Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway. PLoS Pathogens, 11(8), e1005091.

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Molecular Mechanisms of CRAC Channel Regulation

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All plasmids used in this study were sequenced to confirm their identity. Crbn, Ampk, Slo, or HA-Ubiquitin constructs used in this study were described previously^{26 (link)}. Orai1 (MMM1013-202764440), Orai2 (MMM1013-202859855), and Orai3 (MMM1013-202842392) cDNA were purchased from Open Biosystems to construct expression vectors by a PCR-based strategy. Glutamine synthetase (GS) cDNA were synthesized from mRNA of the testis of mice and GS expression vectors were generated. The complete list of all primer sequences used in the study is provided as a Supplementary table. Anti-Flag (Sigma, F1804), anti-HA (Santa Cruz, sc-7392), anti-GST (Santa Cruz, sc-138), anti-Crbn (Sigma, HPA045910), anti-Orai1 (Santa Cruz, sc-68895), anti-Orai1 (Alomone labs, ACC-062), anti-Orai2 (Abcam, ab180146), anti-Ampkα (Cell signaling, #2532), anti-phospho-AMPKα (cell signaling, #2535), anti-Stim1 (Abcam, ab108994), anti-Cul4A (Abcam, ab92554), anti-DDB1 (Bethyl Laboratories, A300-462A), anti-phospho-raptor (Cell signaling, #2083), anti-raptor (Cell signaling, #2280), anti-phospho-S6K (Cell signaling, #9206), anti-S6K (Cell signaling, #9202), anti-Lamin B (Santa Cruz, sc-374015), anti-Erk2 (Santa Cruz, sc-154), anti-CD16/32 (BioLegend, #101302), anti-CD11b (BioLegend, #1010207), and anti-β-Actin (Santa Cruz, sc-1616) were purchased.

Moon H., Min C., Kim G., Kim D., Kim K., Lee S.A., Moon B., Yang S., Lee J., Yang S.J., Cho S.K., Lee G., Lee C.S., Park C.S, & Park D. (2020). Crbn modulates calcium influx by regulating Orai1 during efferocytosis. Nature Communications, 11, 5489.

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Inhibition of PAK4, NAMPT, and mTOR Signaling

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50,000 BON-1 cells or 100,000 QGP-1 were grown in 100 mm petri dishes overnight. The following day, each cell line was treated with specified concentrations of KPT-9274, PF-3758308, Everolimus, FK866, and combinations for 72 hours. A total of 50 μg protein lysates from treated and untreated cells were separated in a 10–12% SDS-PAGE and transferred into a nitrocellulose membrane (GE Healthcare, ThermoFisher Scientific, Waltham, MA, USA). Mouse monoclonal antibodies anti-PAK4 (catalog no. sc-81532), anti-NAMPT (PEBF, catalog no. sc-393510), anti-NAPRT (catalog no. sc-398404), anti-β-TUBULIN (catalog no. sc-5274), anti-β-ACTIN (catalog no. sc-8432) from Santa Cruz Biotechnology, and anti-pmTOR (catalog no. 5536S), anti-mTOR (catalog no. 2972S), anti-RICTOR (catalog no. 9476S), anti-RAPTOR (catalog no. 2280S), anti-pP70S6K (catalog no. 9204S), anti-P70S6K (catalog no. 2708S) from cell signaling technology and were used at a 1:1000 dilution in (3% non-fat milk or 5% BSA) PBS with 0.1% Tween-20 (catalog no. P7949, Sigma-Aldrich, St. Louis, MO, USA).

Mpilla G.B., Uddin M.H., Al-Hallak M.N., Aboukameel A., Li Y., Kim S.H., Beydoun R., Dyson G., Baloglu E., Senapedis W.T., Landesman Y., Wagner K.U., Viola N.T., El-Rayes B.F., Philip P.A., Mohammad R.M, & Azmi A.S. (2021). PAK4-NAMPT Dual Inhibition Sensitizes Pancreatic Neuroendocrine Tumors to Everolimus. Molecular cancer therapeutics, 20(10), 1836-1845.

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mTOR Modulation Impacts Endothelial Angiogenesis

Cited 2 times

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Medium 199 (M199), Hank's Balanced Salt Solution (HBSS), fetal bovine serum (FBS), and endothelial cell growth supplement were purchased from Invitrogen (Burlington, ON). VEGF-A was from Peprotech (Princeton, NJ). Rapamycin, PP242, a highly specific mTOR active-site inhibitor [12 (link)], and anti-tubulin-α was from Millipore (Temecula, CA). Ku-0063794, a second specific mTOR inhibitor [13 (link)] was from Sigma (St. Louis, MO). Anti-Akt1 was from Protein Tech (Chicago, IL). Hiperfect, non-silencing short interfering RNA (siRNA) and Akt1 silencing siRNA were from Qiagen Inc (Mississauga, ON). Human tumor necrosis factor-α (TNFα) was from Cedarlane (Mississauga, ON). Cycloheximide, phalloidin-FITC, anti-vinculin, and DAPI were from Sigma. Anti-S6K was from Abcam (Cambridge, UK). Anti-phospho-Akt^S473, anti-phospho-S6K^T389, anti-FAK, anti-phospho-FAK^Y397, anti-raptor, anti-rictor, and rictor siRNA were from Cell Signaling Technology (Danvers, MA). ON-TARGETplus human raptor siRNA-SMARTpool was from Thermo Scientific (Waltham, MA).

Farhan M.A., Carmine-Simmen K., Lewis J.D., Moore R.B, & Murray A.G. (2015). Endothelial Cell mTOR Complex-2 Regulates Sprouting Angiogenesis. PLoS ONE, 10(8), e0135245.

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Western Blot Analysis of Signaling Proteins

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Primary antibodies used in this study were purchased from Cell Signaling Technology (Beverly, MA): anti-pPKA^Thr198/PKA, anti-pCREB^Ser133/CREB, anti-pSTAT3^Tyr705/STAT3, anti-pSrc^Ser17/Src, anti-pACC ^Ser79/ACC, anti-pAkt^Ser473/Akt, anti-pAMPK ^Thr172/AMPK, anti-p4EBP1^Thr37/46/4EBP1, anti-pmTOR^Ser2448/mTOR, anti-pP70S6K^Thr389/P70S6K, anti-EPAC-1, anti-BEATA2_AR, anti-FAK, anti-FASN, anti-GLUT4, anti-OCT3, anti-NOTCH, anti-PGC1, anti-pPRAS40^Thr246, anti-PRAS40, anti-pRAPTOR^Ser792, anti-RAPTOR, and anti-PI3K110α. Anti-BCL-2 was purchased from BD Bioscience (San Jose, CA). Anti-P27 was purchased from Thermo Fisher Scientific (Waltham, MA). Anti-rabbit immunoglobulin-horseradish peroxidase-conjugated secondary antibody, LumiGLO reagent with peroxide and cAMP assay kit were also purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-IGF1Rα, anti-HMGCR, anti-P21, anti-SCD1, and anti-SCEBP1 were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX); mouse anti-β-actin primary antibody was obtained from Sigma Aldrich (St. Louis, MO). The 1-methyl-1-nitrosourea (MNU) was obtained from Ash Stevens (Detroit, MI) and stored at −80°C prior to use. Metformin and buformin were obtained from Waco Pure Chemical Industries, (Waco, TX); phenformin was obtained from Sigma Aldrich (St. Louis, MO).

Zhu Z., Jiang W., Thompson M.D., Echeverria D., McGinley J.N, & Thompson H.J. (2015). Effects of Metformin, Buformin, and Phenformin on the Post Initiation Stage of Chemically-Induced Mammary Carcinogenesis in the Rat. Cancer prevention research (Philadelphia, Pa.), 8(6), 518-527.

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Protein Modification and Regulation Assays

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Antibodies for Western Blot Analysis

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All primary antibodies used for western blots were purchased from commercial sources. Antibodies from Santa Cruz include: anti-beclin 1 (sc11427), anti-myc HRP (sc40 HRP) and anti-β-actin HRP (sc47778 HRP). Antibodies from Cell Signaling Technology include: anti-UVRAG (#11315), anti-Vps34 (#4263), anti-Atg14 (#96752, to detect mouse Atg14), anti-p-AMPKα(Thr172) (#2535), anti-AMPKα (#2532), anti-p-Raptor(Ser792) (#2083), anti-Raptor (#2280), anti-TBC1D1 (#66433) and anti-HA (#3724, to detect Tlr9-HA in tissue lysates of Tlr9-HA KI mice). Other primary antibodies include: anti-p-ACC(Ser79) (#07–303, Millipore), anti-ACC (#04–322, Millipore), anti-p-TBC1D1(Ser237) (#07–2268, Millipore), anti-Atg14 (M184–3, MBL International, to detect human ATG14), anti-HA high affinity-HRP (#12013819001, Roche, to detect overexpressed TLR9-HA in cells and Tlr9-HA after immunoprecipitation from muscle lysates of the Tlr9-HA KI mice), anti-Flag M2-HRP (A8592, Sigma), and anti-GLUT4 (GT41-A, Alpha Diagnonstic International). To detect beclin 1 in Tlr9-HA immunoprecipitates from muscle lysates by western blot analysis, 10% MINI-PROTEAN TGX Precast gels (Bio-Rad) were used. For detection of all other proteins, 4–20% Precast gels were used.

Liu Y., Nguyen P.T., Wang X., Zhao Y., Meacham C.E., Zou Z., Bordieanu B., Johanns M., Vertommen D., Wijshake T., May H., Xiao G., Shoji-Kawata S., Rider M.H., Morrison S.J., Mishra P, & Levine B. (2020). Tlr9 and Beclin 1 Cross-Talk Regulates Muscle AMPK Activation in Exercise. Nature, 578(7796), 605-609.

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Comprehensive Antibody Collection for Cell Signaling

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Antibodies used in this study included anti-STING (13647; Cell Signaling Technology), anti-Flag M2 (F3165; Sigma-Aldrich), anti-HA (H3663; Sigma-Aldrich), anti-EGFP (GL-8; Clontech), anti-LC3 (7543; Sigma-Aldrich), anti-p62 (PM045; MBL), anti-STX17 (HPA001204; Sigma-Aldrich), anti-SNAP29 antibody (111303, SYSY, or sc-135564; Santa Cruz Biotechnology), anti-LAMP1(L1418; Sigma-Aldrich), anti-LAMP2 (sc-18822; Santa Cruz or L0668; Sigma-Aldrich), anti-Myc (9E10, DSHB), anti-WIPI2 (ab105459; Abcam), anti-FIP200 (17250; ProteinTech), anti-ATG16L (PM040; MBL), anti-α-Tubulin (E7; DSHB), anti-VAMP8 (ab76021; Abcam), anti-GABARAP (ab109364; Abcam), anti-AMPK (2532; Cell Signaling Technology), anti-p-AMPK T172 (2535; Cell Signaling Technology), anti-TBC1D1(66433; Cell Signaling Technology), anti-p-TBC1D1 Ser237(07-2268; Sigma-Aldrich), anti-Raptor (2280; Cell Signaling Technology), anti-p-Raptor Ser792 (2083; Cell Signaling Technology), anti-TSC2 (4308; Cell Signaling Technology), anti-p-TSC2 Ser1387 (5584; Cell Signaling Technology), anti-ACC (3662; Cell Signaling Technology), anti-p-ACC Ser79 (3661; Cell Signaling Technology), anti-Glut4 (GT-41-A; Alpha diagnostic international), anti-Laminin2 (L0663; Sigma-Aldrich), anti-CD36 (80080; Abcam), anti-Dystrophin (15277; Abcam), and LysoTracker (ENZ-51005; Enzo).

Rong Y., Zhang S., Nandi N., Wu Z., Li L., Liu Y., Wei Y., Zhao Y., Yuan W., Zhou C., Xiao G., Levine B., Yan N., Mou S., Deng L., Tang Z., Liu X., Kramer H, & Zhong Q. (2022). STING controls energy stress-induced autophagy and energy metabolism via STX17. The Journal of Cell Biology, 221(7), e202202060.

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