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Pfoxo1 3a

Manufactured by Cell Signaling Technology
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PFoxo1/3a is a lab equipment product offered by Cell Signaling Technology. It is an antibody that specifically recognizes phosphorylated forms of the transcription factors FOXO1 and FOXO3a.

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Lab products found in correlation

Pakt s473, by Cell Signaling Technology (2 mentions) H2dcfda, by Thermo Fisher Scientific (2 mentions) Complete ultra tablet, by Roche (1 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions) P akt, by Cell Signaling Technology (1 mentions) Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions) P erk, by Cell Signaling Technology (1 mentions) β actin, by Santa Cruz Biotechnology (1 mentions) Pan akt, by Cell Signaling Technology (1 mentions) Ps473, by Cell Signaling Technology (1 mentions) Alexa fluor 647 coupled antibody against γh2a x protein, by BD (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Smad2 3, by Cell Signaling Technology (1 mentions) Sb505124, by Selleck Chemicals (1 mentions) Sc 373910, by Santa Cruz Biotechnology (1 mentions) P smad2 3, by Cell Signaling Technology (1 mentions) Snail, by Cell Signaling Technology (1 mentions) Foxo3a, by Cell Signaling Technology (1 mentions) P foxo3a, by Cell Signaling Technology (1 mentions) N cadherin, by Cell Signaling Technology (1 mentions)

5 protocols using pfoxo1 3a

1

Western Blot Analysis of Stimulated T and B Cells

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Purified T and B cells were isolated from murine spleens and stimulated as described above (PIP3 measurement). For performing western blots the stimulation period was 5 min. Isolated T cells were stimulated with anti-CD3 1 µg/mL and anti-CD28 (2 µg/mL) with or without 10 Nm nemiralisb; or B cells with anti-IgM 4 µg/mL (AffinPure F(ab’)2 Fragment goat anti-mouse IgM from Jackson ImmunosResearch labs).
Stimulated T and B cells were lysed with ice-cold lysis buffer (50 mM HEPES, 150 mM NaCl, 10 mM NaF, 10 mM Indoacetamide, 1% IGEPAL and proteinase inhibitors (Complete Ultra tablets, Roche)) for 15–20 min. Lysates were centrifuged at 15,000×g for 10 min at 4 °C and supernatants were mixed with NuPage LDS sample buffer (life technologies). Samples were heated at 70 °C and resolved on 4–12% NuPage bis-tris gel (Invitrogen), transferred to PVDF membranes and probed with the following antibodies: pAKT (T308, Cell Signaling, 1 in 1000 dilution); total AKT1 (2H10, Cell signaling, 1 in 2000 dilution); p110δ (Sc7176, Santa Cruz Biotechnology, 1 in 200 dilution); pS6 (S235/236, Cell Signaling, 1 in 500 dilution); pFoxo1/3a (T24/T32, Cell Signaling, 1 in 1000 dilution); pErk (p44/42, Cell Signaling, 1 in 200 dilution); βActin (Sc47778, Santa Cruz Biotechnology, 1 in 2000 dilution).
Stark A.K., Chandra A., Chakraborty K., Alam R., Carbonaro V., Clark J., Sriskantharajah S., Bradley G., Richter A.G., Banham-Hall E., Clatworthy M.R., Nejentsev S., Hamblin J.N., Hessel E.M., Condliffe A.M, & Okkenhaug K. (2018). PI3Kδ hyper-activation promotes development of B cells that exacerbate Streptococcus pneumoniae infection in an antibody-independent manner. Nature Communications, 9, 3174.
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2

Akt Inhibitor MK-2206 Protocol

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The MK-2206 Akt-inhibitor was purchased from Selleckchem (Houston, TX, USA). The mouse monoclonal ß-Actin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA). panAkt, pS473, pT308, panMERIT40, pMERIT40, pFOXO1/3a, panFOXO1, eGFP and ß-Actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Alexa Fluor 647-coupled antibody against γH2A.X protein was obtained from Becton Dickinson (Heidelberg, Germany). Hoechst33342 was purchased from Invitrogen (Eugene, OR, USA). DAKO Fluorescent mounting medium from Dako North America Inc. (Carpinteria, CA, USA) was used. All other chemicals were acquired from Sigma-Aldrich (St. Louis, MO, USA).
Szymonowicz K., Oeck S., Krysztofiak A., van der Linden J., Iliakis G, & Jendrossek V. (2018). Restraining Akt1 Phosphorylation Attenuates the Repair of Radiation-Induced DNA Double-Strand Breaks and Reduces the Survival of Irradiated Cancer Cells. International Journal of Molecular Sciences, 19(8), 2233.
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3

Akt1 Knockdown Effects on EMT Markers

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Human PC3 and DU145 cell lines were purchased from ATCC (Manassas, VA), cultured in DMEM high glucose medium (Hyclone, Logan, UT) with 10% Fetal bovine serum (FBS, Atlanta Biologicals, Atlanta, GA), 100 U/ml penicillin, and 100 mg/ml streptomycin in a humidified incubator at 37°C and 5% CO2. Cells were routinely passaged and when they were 80–90% confluent, transfection was carried out using SMARTvector 2.0 Lentivirus ShControl (non-targeting) and ShAkt1 (ACGCTTAACCTTTCCGCTG) from Dharmacon (Lafayette, CO), followed by selection with puromycin (0.6 ng/ml, Sigma Millipore, St. Louis, MO). Primary antibodies against Akt1 (Cat #2938), pSer473Akt (Cat #4060), pThr308Akt (Cat #2965), pFoxO1/3a (Cat #9464), pFoxO3a (Cat #9465), FoxO3a (Cat #2497), FoxO1 (Cat #2880), pSmad2/3 (Cat #8828), Smad2/3 (Cat #8685), Snail (Cat #3879), E-cadherin (Cat #3195), and N-cadherin (Cat #4061) were purchased from Cell Signaling (Danvers, MA). Nodal antibodies (Cat # SC-28913 and SC-373910) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). β-actin antibody (Cat #A5441) was purchased from Sigma (St. Louis, MO). Triciribine (TCBN) and SB505124 were purchased from Selleckchem (Houston, TX), and FoxO1/3a inhibitor (AS1842856) was purchased from Calbiochem (San Diego, CA). All other reagents were purchased from Fisher Scientific (Hanover Park, IL).
Alwhaibi A., Verma A., Artham S., Adil M.S, & Somanath P.R. (2019). Nodal pathway activation due to Akt1 suppression is a molecular switch for prostate cancer cell epithelial-to-mesenchymal transition and metastasis. Biochemical pharmacology, 168, 1-13.
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4

Immunoblotting analysis of signaling proteins

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Whole-cell lysates were prepared and subjected to immunoblotting as previously described38 (link) using antibodies against BCL-3 (sc-185), NF-κB1 (p105/p50) (sc-8414; Santa Cruz Biotechnology, California, USA), p-AKT S473 (4058) and T308 (4056), total AKT (9272), p-FOXO1/3a (9464), p-GSK-3α/β (9331; Cell Signalling Technology, Massachusetts, USA), NF-κB2 (p100/p52) (05-361; EMD Millipore), HIF-1α (610958; BD Biosciences, UK). Equal loading was confirmed with α-tubulin (T9026; Sigma). Blots were quantified using ImageJ software (RSB, NIH, Maryland, USA).
Urban B.C., Collard T.J., Eagle C.J., Southern S.L., Greenhough A., Hamdollah-Zadeh M., Ghosh A., Poulsom R., Paraskeva C., Silver A, & Williams A.C. (2015). BCL-3 expression promotes colorectal tumorigenesis through activation of AKT signalling. Gut, 65(7), 1151-1164.
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5

Multimodal Immune Profiling Protocol

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B16-F10 and LLC were obtained from ATCC. MC38 was obtained from Dario Vignali. B16OVA (MO5) was obtained from Per Basse and Lou Falo. OVA-ex-pressing Vaccinia virus was originally generated by Yewdell and Bennink and obtained from Jonathan Powell. Most antibodies for flow cytometry were obtained from BioLegend. MitoTracker Green FM, MitoTracker Deep Red FM, tetramethylrhodamine ester (TMRE), and H2-DCFDA were obtained from ThermoFisher. VDAC antibody was obtained from Abcam. LC3B, pAktS473, pFoxo1/3a antibodies were obtained from Cell Signaling Technologies and detected after surface staining with simultaneous fixation and permabilization in 1.5% PFA madeup in 1X Permeabilization buffer (eBioscience). 2-NBD-glucose, m-divi-1, and Akt inhibitor VIII were purchased from Cayman Chemical. PGC1α antibody (H-300) was obtained from Santa Cruz Biotechnology, and was detected using the Foxp3 Fix/Perm kit (eBioscience) and Alexa Fluor 647 or Alexa Fluor 488-conjugated anti-rabbit immunoglobulin G (IgG) (Jackson Immunoresearch). Anti-PD-1 blocking antibody (J43) and its hamster IgG control were obtained from Bio-X-Cell. CellTrace Violet was purchased from eBioscience, and CFSE was purchased from BioLegend.
Scharping N.E., Menk A.V., Moreci R.S., Whetstone R.D., Dadey R.E., Watkins S.C., Ferris R.L, & Delgoffe G.M. (2016). The Tumor Microenvironment Represses T Cell Mitochondrial Biogenesis to Drive Intratumoral T Cell Metabolic Insufficiency and Dysfunction. Immunity, 45(2), 374-388.
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