Ppdk1 s241

Manufactured by Cell Signaling Technology

Sourced in United States

PPDK1 S241 is a laboratory equipment product that measures the phosphorylation of the PPDK1 protein at the serine 241 residue. This product is used for research purposes to quantify the phosphorylation level of this specific site on the PPDK1 protein.

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8 protocols using ppdk1 s241

Immunoblotting for Protein Signaling Pathways

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Equal amounts of proteins of whole cell lysates or nuclear extracts [36 (link)] were separated by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride (PVD) membrane, probed with specific antibodies, and detected using ECL detection reagent (GE Healthcare).
Antibodies against p-PDK1^S241 (#3031), p-GSK-3β^S9 (#9336), p-BAD^S136 (#39295), AKT1 (#2967), and β-catenin (#9562) were from Cell Signaling. P-Akt1^S473 (#05-736) antibody was from Upstate (Charlottesville, VA), p65 (1546-1), p-Akt1^T308 (#2214-1), and p-P70S6K^T389 (#1175-1) were supplied by Epitomics (Burlingame, CA), and c-myc (M5546) was from Sigma. The antibodies against IκBα (sc-371), P70S6K (sc-8418), BAD (sc-8044), GSK-3 (sc-7291), and topoisomerase I (TOPO I) (sc-10783) were from Santa Cruz Biotechnology (Santa Cruz, CA), PDK1 (IMG-30048) was from Imgenex (San Diego, CA), and actin (MAB1501) was from Chemicon (Temecula, CA).

Schmidt C., Loos C., Jin L., Schmiech M., Schmidt C.Q., Gaafary M.E., Syrovets T, & Simmet T. (2017). Acetyl-lupeolic acid inhibits Akt signaling and induces apoptosis in chemoresistant prostate cancer cells in vitro and in vivo. Oncotarget, 8(33), 55147-55161.

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Analyzing Protein Signaling Cascades with Western Blotting

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Westerns were as described^{25 (link)}. Antibodies used in Westerns: anti- pAKT S473, AKT, pPDK1 S241, PDK1, p STAT3 Y705 and STAT3 were from Cell Signaling; for E-Cadherin, N-Cadherin and p27 from Transduction Labs and for p27pT157 and p27pT198 from R&D Systems. p27 immunoprecipitation used PAb C-19 from Santa Cruz Biotechnology.

Zhao D., Besser A.H., Wander S.A., Sun J., Zhou W., Wang B., Ince T., Durante M.A., Guo W., Mills G., Theodorescu D, & Slingerland J. (2015). Cytoplasmic p27 promotes epithelial–mesenchymal transition and tumor metastasis via STAT3-mediated Twist1 upregulation. Oncogene, 34(43), 5447-5459.

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Investigating Akt Signaling Pathways

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Western blots were performed as described (Yang et al., 2007 (link)) with the following antibodies: p-Akt (S473), p-Akt (T308), Akt, p-p70S6K (T389), p70S6K, RSK1, p-RSK2 (S227), RSK2, p-PDK1 (S241), PDK1, PI3K p110α, and GAPDH (Cell Signaling Technology Inc., MA, USA); Na⁺/K⁺-ATPase α1 (Abcam Inc. Cambridge, UK); and an antibody against TGEV N protein, as described (Yang et al., 2007 (link)). Ouabain (O3125, ≧95%, HPLC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). p-RSK1 (T359/S363) antibody, BX795, rottlerin, GF109203X, rapamycin, and triciribine were purchased from Merck Millipore Calbiochem (Billerica, MA). LY294002, PP2, KX2-391 and SU6656 were from Selleckchem (Houston, TX, USA). Farnesyl thiosalicylic acid (FTA, a Rac1 inhibitor) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Wortmannin was purchased from Life Technologies (San Diego, CA, USA).

Yang C.W., Chang H.Y., Lee Y.Z., Hsu H.Y, & Lee S.J. (2018). The cardenolide ouabain suppresses coronaviral replication via augmenting a Na+/K+-ATPase-dependent PI3K_PDK1 axis signaling. Toxicology and Applied Pharmacology, 356, 90-97.

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Immunoblotting and Immunohistochemistry for Protein Analysis

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Protein lysates of cells growing as a monolayer were prepared as described previously [40 (link)]. Protein concentrations were determined by the Bradford assay (Biorad). 30 μg of protein were loaded on a 4–12% Bis-Tris gel (Invitrogen) and blotted onto a nitrocellulose membrane.
Immunohistochemistry of paraffin-embedded sections was performed as described previously [45 (link)]. Antigen-retrieval consisted of microwaving in 0.01 M citrate buffer (pH 6.0) for 10 min. Immunoperoxidase-based detection was performed using the Histostain-Plus 3rd Gen IHC Detection Kit (Invitrogen/Thermo Fisher Scientific) according to manufacturer's recommendations. Cells were analyzed using an Olympus AX70 epifluorescence microscope equipped with a SpotRT digital camera.
Primary antibodies used for immunoblotting and immunohistochemistry were ABL1, CDK2, pTyr (all Santa Cruz), actin (Sigma), pABL1 Y412, pAKT S473, AKT, pCDK2 T160, cleaved caspase 3, pCRKL Y207, CRKL, pKIT Y719, pMAPK p42/44 T202, pPDK1 S241, PDK1, PP2A, pS6K T389, S6K (all Cell Signaling Technologies), CIP2A, PHLPP, SET (all Bethyl Laboratories), cyclin A (Novocastra), KIT (DakoCytomation) and MAPK (Invitrogen/Thermo Fisher Scientific).

Rausch J.L., Boichuk S., Ali A.A., Patil S.S., Lee D.M., Brown M.F., Makielski K.R., Liu Y., Taguchi T., Kuan S.F, & Duensing A. (2016). Opposing roles of KIT and ABL1 in the therapeutic response of gastrointestinal stromal tumor (GIST) cells to imatinib mesylate. Oncotarget, 8(3), 4471-4483.

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Western Blot Analysis of Signaling Proteins

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The following antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA) : EGFR (#2232), pEGFR Y1068 (#2234), HER3 (#4754), pHER3 Y1197 (#4561), pBRAF S445 (#2696), MEK (#9126), pMEK S217/221 (#9154), ERK (#9102), pERK T202/Y204 (#9101), PDK1 (#3062), pPDK1 S241 (#3061), AKT (#9272), pAKT S473 (#9271), pAKT T308 (#9275), ribosomal protein S6 (#2317), pS6 S240/S244 (#5364), FAK (#3285), pFAK Y397 (#8556), SFKs (#2108), pSFKs Y416 (#2101), YES (#3201), Integrin Antibody Sampler Kit (#4749), PARP (#9542), E-cadherin (#3195), Vimentin (#3932), horseradish peroxidase (HRP)-conjugated anti-mouse (#7076) and HRP-conjugated anti-rabbit (#7074). The actin antibody (A2066) was purchased from Sigma-Aldrich. The BRAF antibody (sc-55522) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). For immunoblot, cells were harvested, washed in PBS, and lysed in RIPA buffer [50 mM Tris•HCl (pH 8.0), 150 mM sodium chloride, 5 mM magnesium chloride, 1% Triton X-100. 0.5% sodium deoxycholate, 0.1% SDS, 40 mM sodium fluoride, 1 mM sodium orthovanadate, and complete protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA)]. Western Lightning ECL reagent (Perkin-Elmer) as used for signal detection. Phosphorylated bands were quantified using ImageJ software.

Ichihara E., Westover D., Meador C.B., Yan Y., Bauer J.A., Lu P., Ye F., Kulick A., de Stanchina E., McEwen R., Ladanyi M., Cross D., Pao W, & Lovly C.M. (2017). SFK/FAK signaling attenuates osimertinib efficacy in both drug-sensitive and drug-resistant models of EGFR-mutant lung cancer. Cancer research, 77(11), 2990-3000.

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Western Blot Analysis of Cell Signaling Proteins

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Western blot was performed on whole-cell extracts as previously described [61 (link)] using the antibodies mentioned. FOXM1 (c-20; sc-502), P-PERK (Thr 981) (sc-32577), p27^Kip1 (sc-528) and β-tubulin H-235; sc-9104) antibodies were purchased from Santa Cruz Biotechnology (CA, USA). The P-FOXO1 (Thr24)/FOXO3 (Thr32) (CST# 9464) and FOXO3 (CST#2497), PERK (CST#3192), FOXO1 (CST#9454), FOXO4 (CST #9472), Bim (CST#2933), P-AKT (S473) (CST#9271), P-AKT(T308) (CST#9275), AKT (CST#9272) P-PDK-1 (S241) (CST#3061), PDK-1 (CST#3062) and eIF2α (CST#5324) were purchased from Cell Signaling Technology (New England Biolabs Ltd. Hitchin, UK). The P-elF2α antibody (ab32157; Abcam; Cambridge, UK). The primary antibodies (1:1000) were detected using horseradish peroxidase-conjugated secondary antibody (1:2000, DAKO, Glostrup, Denmark) and visualised using the ECL detection system (PERKinElmer Ltd, Beaconsfield, UK).

Alasiri G., Jiramongkol Y., Zona S., Fan L.Y., Mahmud Z., Gong G., Lee H.J, & Lam E.W. (2019). Regulation of PERK expression by FOXO3: a vulnerability of drug-resistant cancer cells. Oncogene, 38(36), 6382-6398.

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Antibody-based Signaling Pathway Analysis

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Antibodies of COL3A1 (13548-1-AP), cyclin D1 (60186-1-Ig) and PI3K (20583-1-AP) were purchased from Proteintech (Wuhan, Hubei, China). GAPDH antibody (CW0100) was bought from CWBIO, Beijing, China. Other antibodies were purchased from Cell Signaling Technology (CST): PDK1 (#3062), p-PDK1S241 (#3061), p-AktS473 (D9E, #4060), p-AktT308 (244F9, 4056), p-p70S6KT389 (108D2, #9234), p70 S6 Kinase (49D7, #2708), p-mTORS2448 (#2971), mTOR (#2972), p-GSK-3α/βS21/9 (D17D2, #8566) and GSK-3α/β (D75D3,#5676).

Wang X.Q., Tang Z.X., Yu D., Cui S.J., Jiang Y.H., Zhang Q., Wang J., Yang P.Y, & Liu F. (2016). Epithelial but not stromal expression of collagen alpha-1(III) is a diagnostic and prognostic indicator of colorectal carcinoma. Oncotarget, 7(8), 8823-8838.

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Western Blot Analysis of AKT, STAT3, and p27 Signaling

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Western blots were performed as described.^{25 (link)} Antibodies used in western blots were as follows: anti-pAKT S473, AKT, pPDK1 S241, PDK1, p STAT3 Y705 and STAT3 (from Cell Signaling); for E-cadherin, N-cadherin and p27 from Transduction Labs; and for p27pT157 and p27pT198 from R&D Systems. p27 immunoprecipitation used PAb C-19 from Santa Cruz Biotechnology.

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