P raptor

Manufactured by Cell Signaling Technology

Sourced in United States

The P-Raptor is a laboratory equipment product offered by Cell Signaling Technology. It is designed to facilitate the analysis of protein phosphorylation, a key cellular signaling process. The P-Raptor provides a platform for the detection and quantification of phosphorylated proteins, enabling researchers to study signal transduction pathways and cellular responses.

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Lab products found in correlation

Close spelling variants of this product

Raptor (86 citations) Anti-p-Raptor(Ser792) (4 citations) P‐Raptor‐S792 (2 citations)

19 protocols using p raptor

Investigating Estrogen Receptor Signaling

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Western blot analysis used specific antibodies for ERα (HC-20, Santa Cruz); ERK2 (D-2, Santa Cruz); and pS6, pS6K, pmTOR, pRAPTOR, pRICTOR, pMAPK (Cell Signaling). Coimmunoprecipitation assays used antibodies for SRC3 (Santa Cruz, C-20) and ERα (Santa Cruz, F10). ChIP assays were carried out as described (9 (link), 11 (link)). Antibodies used were for ERα (HC20), ERK2 (Santa Cruz, D2 and C14), and pSer⁵ RNA Pol II (Santa Cruz, sc-47701). ChIP DNA was isolated using QIAGEN PCR purification kit and used for ChIP-seq analysis and quantitative real-time PCR (qPCR). qPCR was used to calculate recruitment to the regions studied, as described (9 (link)).

Madak-Erdogan Z., Kim S.H., Gong P., Zhao Y.C., Zhang H., Chambliss K.L., Carlson K.E., Mayne C.G., Shaul P.W., Korach K.S., Katzenellenbogen J.A, & Katzenellenbogen B.S. (2016). Design of pathway-preferential estrogens that provide beneficial metabolic and vascular effects without stimulating reproductive tissues. Science signaling, 9(429), ra53.

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Isolation and Analysis of Cellular Fractions

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Nuclear and cytoplasmic extracts from naive T cells cultured for 3 days were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce), as previously described^{21 (link)}. The purity of the nuclear and cytoplasmic fractions was verified by probing with antibodies against Lamin B1 (D4Q4Z; 1,000×; Cell Signaling Technology). Whole-cell extracts were immunoprecipitated with an anti-Raptor (24C12; 100×; Cell Signaling Technology), anti-Def6 (Rabbit polyclonal; 100×^{21 (link)}), anti-p62 (H-290; 50×; Santa Cruz), anti-TRAF6 (H274; 50×; Santa Cruz), or anti-HA (3F10; 50×; Roche Applied Science) antibodies. Antibodies to p-STAT3 (Y705; 1,000×), p-4E-BP (T37/46; 1,000×), 4E-BP (1,000×), p-S6K1 (S371; 1,000×), S6K1 (1,000×), p-AKT (S473; 1,000×), AKT (1,000×), p-AKT (T308; 1,000×), p-PRAS40 (T246; 1,000×), PRAS-40 (1,000×), p-AMPK (T172; 1,000×), AMPK (1,000×), p-Raptor (S792; 1,000×) and p62 (5114; 1,000×) were obtained from Cell Signaling Technology. Antibodies to IRF4 (M-17; 1,000×), TRAF6 (H274; 500×), and c-Myc (9E10; 500×) were obtained from Santa Cruz. Anti-Bcl6 antibody was obtained from BD (K112-91; 1,000×). Anti-Flag monoclonal antibody M2 (horseradish peroxidase (HRP)) was obtained from Sigma (1,000×).

Yi W., Gupta S., Ricker E., Manni M., Jessberger R., Chinenov Y., Molina H, & Pernis A.B. (2017). The mTORC1-4E-BP-eIF4E axis controls de novo Bcl6 protein synthesis in T cells and systemic autoimmunity. Nature Communications, 8, 254.

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Western Blotting Analysis of Frozen Tissues

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Western blotting was performed on snap-frozen tissue samples (SED: n = 3; EX: n = 3; EX + DOX: n = 3). Tissue lysates were prepared by homogenization and the protein concentration was assessed as previously described (Kolwicz et al. 2009 (link)). Equal amounts of protein (50 μg) were separated by SDS-PAGE and transferred to nitrocellulose membrane (BioRad, Hercules, CA). Primary antibodies for: phosphorylated (p)-AMPK at threonine(thr)172, AMPK, p-Akt at serine (ser) 473, Akt, p-TSC2(ser1387), p-TSC2(thr1462), TSC2, p-mTOR(ser2481), p-mTOR(ser2448), mTOR, p-Raptor(ser792), Raptor, p-p70S6K(thr387), p70S6K, p-ULK1(ser317), p-ULK1(ser757),and ULK1 (Cell Signaling, Danvers, MA) were used. Signals were visualized by enhanced chemiluminescence, digitized, and quantified with Image J software (NIH, Bethesda, MD).

Sturgeon K., Muthukumaran G., Ding D., Bajulaiye A., Ferrari V, & Libonati J.R. (2015). Moderate-intensity treadmill exercise training decreases murine cardiomyocyte cross-sectional area. Physiological Reports, 3(5), e12406.

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Compound C and Sodium Palmitate Signaling Pathway Analysis

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Compound C was obtained from Enzo Life Sciences. Sodium Palmitate is from NU-CHEK Prep Inc. For western blotting, anti-Flag M2 (RRID:AB_259529) antibody was purchased from Sigma, antibodies against SGK1 (RRID:AB_2687476), SGK2 (RRID:AB_10828732), SGK3 (RRID:AB_10949507), HSP90 (RRID:AB_2233307), Akt (RRID:AB_915783), p-Akt (Thr308) (RRID:AB_2255933), p-Akt (Ser473) (RRID:AB_2315049), NDRG1 (RRID: AB_11140640), p-NDRG1(Thr346) (RRID:AB_10693451), FoxO1 (RRID:AB_2106495), p-FoxO1/3 (Thr24/32) (RRID:AB_2106814), S6K (RRID:AB_390722), p-S6K (Thr389) (RRID:AB_2269803), S6 (RRID:AB_331355), p-S6 (S240/244) (RRID:AB_10694233), 4EBP1 (RRID:AB_2097841), p-4EBP1 (Thr37/46) (RRID:AB_560835), p-4EBP1 (Ser65) (RRID:AB_330947), AMPKα (RRID:AB_10624867), p-AMPKα (Thr172) (RRID:AB_331250), p-AMPKα(Ser485/491) (RRID:AB_331250), ACC1 (RRID:AB_2219397), p-ACC1 (Ser79) (RRID:AB_330337), Raptor (RRID:AB_561245)and p-Raptor (Ser792) (RRID:AB_2249475) were obtained from Cell Signaling Technology. Anti-actin (C4) was obtained from Abcam.

Zhou B., Zhang Y., Li S., Wu L., Fejes-Toth G., Naray-Fejes-Toth A, & Soukas A.A. (2021). Serum- and glucocorticoid-induced kinase drives hepatic insulin resistance by directly inhibiting AMP-activated protein kinase. Cell reports, 37(1), 109785.

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Quantitative Immunoblotting of AMPK Signaling

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Primary antibodies were used in the indicated dilutions: PARP (#9542, Cell Signaling) 1:1000, α-Tubulin (#B-5-1-2, Santa Cruz) 1:2500, pACC (#3661, Cell Signaling) 1:2000, tACC (#3676, Cell Signaling) 1:2000, pRAPTOR (#2083, Cell Signaling) 1:1000, tRAPTOR (#2280, Cell Signaling) 1:1000, p AMPKα1α2 (#2531, Cell Signaling) 1:1000, AMPKα1α2 (#2532, Cell Signaling) 1:1000, AMPKα2 (#AF2850, R&D systems) 1:1000, and AMPKα1 (#2795, Cell Signaling) 1:1000. Anti-mouse, and anti-rabbit secondary antibodies conjugated to Alexa Fluor 680 (Invitrogen, Carlsbad, CA) or IRDye800, and IRDye680LT were used at 1:5000-1:10,000 dilutions. Additional reagents included recombinant AMPKα1β1γ1 (#P47-10H, SignalChem) and AMPKα2β1γ1 (#P48-10H, SignalChem), SAMS peptide (S07-58, Cquential Solutions), and radioactive α-³²P-ATP (64014, MPBIo).

Das B., Neilsen B.K., Fisher K.W., Gehring D., Hu Y., Volle D.J., Kim H.S., McCall J.L., Kelly D.L., MacMillan J.B., White M.A, & Lewis R.E. (2018). A Functional Signature Ontology (FUSION) screen detects an AMPK inhibitor with selective toxicity toward human colon tumor cells. Scientific Reports, 8, 3770.

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8-Cl-Ado Regulation of AMPK and mTOR Pathways

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Exponentially growing cells were treated with 10 μM 8-Cl-Ado for various amounts of time and protein lysates were isolated and analyzed using an Odyssey Infrared Imaging System (LI-COR Biosciences) as described [19 (link)]. Primary antibodies were rabbit polyclonal antibodies against p-AMPKα (Thr172), AMPKa, p-acetyl-coA carboxylase (ACC) (Ser79), p-raptor (Ser792), p-mTOR (Ser2481), p4E-BP1 (Ser65) (Cell Signaling Technology), LC3B, beclin 1 (Novus Biologicals, Inc, Littleton, CO), p62 (Enzo Life Sciences, Farmingdale, NY); rabbit monoclonal antibodies against p-ULK1 (Ser555) clone D1H4, raptor clone 24C12, mTOR clone 7C10 (Cell Signaling Technology), ATG7 (Novus Biologicals, Inc, Littleton, CO); mouse monoclonal antibodies GAPDH clone 6C6 (Abcam, Inc, Cambridge, MA); and goat polyclonal antibody 4E-BP1 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA).

Stellrecht C.M., Vangapandu H.V., Le X.F., Mao W, & Shentu S. (2014). ATP directed agent, 8-chloro-adenosine, induces AMP activated protein kinase activity, leading to autophagic cell death in breast cancer cells. Journal of Hematology & Oncology, 7, 23.

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Inhibitors for mTOR Signaling Pathway

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Monoclonal antibodies against mTOR (Cat No. 29883), p-mTOR (Cat No. 5536), Beclin1 (Cat No. 3956), AMPK (Cat No. 5831), p-AMPK (Cat No. 50081), Raptor (Cat No. 2280), p-Raptor (Cat No. 2083) and LC3 (Cat No. 12741) were purchased from Cell Signaling Technology (CST, MA, USA). PFTα (a p53 inhibitor, Cat No.), Compound C (an AMPK inhibitor, Cat No. 171261), LY294002 (a PI3K/Akt inhibitor, Cat No. L9908), and PD98059 (an MAPK/ERK1/2 inhibitor, Cat No. P215) were purchased from Sigma–Aldrich (USA).

Zhang X., Ma P., Shao T., Xiong Y., Du Q., Chen S., Miao B., Zhang X., Wang X., Huang Y, & Tong D. (2022). Porcine parvovirus triggers autophagy through the AMPK/Raptor/mTOR pathway to promote viral replication in porcine placental trophoblasts. Veterinary Research, 53, 33.

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Autophagy Regulation by G9a and Rheb Signaling

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BIX-01294 (S8006) was purchased from Selleckchem, 3-methyadenine (3-MA, 189490) and Bafilomycin A1 (BAFA1, 196000) were purchased from EMD Millipore. Chloroquine (CQ, C6628) was from Sigma. Compound C (ab120843) and AICAR (ab120358) were purchased from Abcam. Lipofectamine 2000 reagent was purchased from Invitrogen. RIPA buffer was purchased from Cell Signaling Technology (CST), protease inhibitor and phosphatase inhibitor were from Roche. BCA qualification system was purchased from Pierce. Primary antibodies against LC-3 I/II, ATG3, ATG5, ATG7, p-Raptor (Ser792), Raptor, mTOR, p-mTOR(Ser2448), p-ACC (Ser79), p-AMPK α (Thr172), AMPK α, p-S6K (Thr389), p-4E-BP1 (Thr37/46), histone 3, Rheb, β-actin and peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology (CST), p62 was from Novus, H3k9me2 was from Abcam. PVDF membrane was purchased from Bio-rad. The shG9a #1 and shG9a #2 plasmids and a scrambled RNA which used as shcontrol were purchased from GenePharma, the target sequence was shown in S1 Table. GV230-Rheb plasmid was constructed by GeneChem (Gene accession NM_005614), and empty GV230 vector was used as the control. Neromycin was used to screen steady cloning The mRFP-EGFP-LC-3 reporter plasmid (ptfLC-3) was a gift from Tamotsu Yoshimori (Addgene plasmid # 21074)[21 (link)].

Li F., Zeng J., Gao Y., Guan Z., Ma Z., Shi Q., Du C., Jia J., Xu S., Wang X., Chang L., He D, & Guo P. (2015). G9a Inhibition Induces Autophagic Cell Death via AMPK/mTOR Pathway in Bladder Transitional Cell Carcinoma. PLoS ONE, 10(9), e0138390.

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Signaling Pathway Protein Analysis

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Antibodies against Atg5 (2630), p-AKT (4060), AKT (4685), p-mTOR (5536), mTOR (2983), p-S6 (4858), S6 (2317), p-4E-BP1 (2855), 4E-BP1 (9644), p-AMPK (2535), AMPK (5831), p-TSC-2 (3614), TSC-2 (4308), p-Raptor (2083), Raptor (2280), p-Atg13 (26839), Atg13 (13273), ULK1 (8054), p-ULK1 (Ser757) (14202), p-ULK1 (Ser555) (5869), VPS15 (14580), VPS34 (4263), Beclin1 (3495), p-Beclin1 (84966), Atg14 (96752), Atg16L1 (8089), p62 (16177), and GAPDH (5174) were obtained from Cell Signaling Technology (Danvers, MA). Anti-LC3 antibody (L8918) was obtained from Sigma-Aldrich. Anti-Lamp2b antibody (66301-1-Ig) was purchased from Proteintech (Chicago, IL). Horseradish peroxidase (HRP)-conjugated secondary antibodies (7074 or 7076), Alexa Fluor 647-conjugated secondary antibodies (4414), and Alexa Fluor 488-conjugated secondary antibodies (4408) were purchased from Cell Signaling Technology.

Shang C., Zhuang X., Zhang H., Li Y., Zhu Y., Lu J., Ge C., Cong J., Li T., Li N., Tian M., Jin N, & Li X. (2021). Inhibition of Autophagy Suppresses SARS-CoV-2 Replication and Ameliorates Pneumonia in hACE2 Transgenic Mice and Xenografted Human Lung Tissues. Journal of Virology, 95(24), e01537-21.

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Immunohistochemical and Immunoblotting Analyses of Inflammatory Signaling Pathways

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The primary antibodies that were used for immunohistochemistry and immunoblotting included rabbit anti-mouse COX-2 from Cayman Chemicals, cyclin D1 and c-Myc from Santa Cruz Biotechnology; rat anti-mouse F4/80 (marker of macrophages/dendritic cells) from AbD Serotec; rabbit anti-p-ERK, p-p70 S6K (Thr389), p-PI3K p85 (Tyr458), p-PDK1 (Ser241), p-AKT (Thr308), p-mTOR (Ser2448), p-raptor (Ser792), p-elF-4B(Ser422), p-S6 ribosomal protein (p-rpS6, Ser240/244), and rabbit anti-cleaved caspase-3 (9661) from Cell Signaling Technology; rabbit anti-iNOS, rabbit-anti-Ki67 (ab15580), and goat anti-arginase 1 from Abcam, mouse anti-interleukin 4 receptor α (IL-4Rα) and mannose receptor (MR, CD206) from R&D.

Chang J., Vacher J., Yao B., Fan X., Zhang B., Harris R.C, & Zhang M.Z. (2015). Prostaglandin E receptor 4 (EP4) promotes colonic tumorigenesis. Oncotarget, 6(32), 33500-33511.

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