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Pakt t308

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PAKT T308 is a monoclonal antibody that specifically recognizes phosphorylation of Akt (also known as Protein Kinase B) at Threonine 308. Akt is a key regulator of cell growth, proliferation, and survival signaling pathways.

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Lab products found in correlation

Pakt s473, by Cell Signaling Technology (81 mentions) Gapdh, by Cell Signaling Technology (15 mentions) P erk, by Cell Signaling Technology (12 mentions) Total akt, by Cell Signaling Technology (12 mentions) P gsk3β s9, by Cell Signaling Technology (10 mentions) Ripa buffer, by Thermo Fisher Scientific (10 mentions) Ps6 s235 236, by Cell Signaling Technology (9 mentions) 4e bp1, by Cell Signaling Technology (9 mentions) P erk1 2, by Cell Signaling Technology (9 mentions) Cleaved caspase 3, by Cell Signaling Technology (8 mentions) Erk1 2, by Cell Signaling Technology (8 mentions) Pan akt, by Cell Signaling Technology (8 mentions) Ps6 s240 244, by Cell Signaling Technology (7 mentions) Gsk3β, by Cell Signaling Technology (7 mentions) Actin, by Merck Group (6 mentions) P70s6k, by Cell Signaling Technology (6 mentions) β actin, by Cell Signaling Technology (6 mentions) P erk1 2 t202 y204, by Cell Signaling Technology (5 mentions) P 4e bp1 t37 46, by Cell Signaling Technology (5 mentions) P 4ebp1 s65, by Cell Signaling Technology (5 mentions)

118 protocols using pakt t308

1

Immunoblotting Analysis of Signaling Proteins

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The cultured cells were washed and lysed in cell extraction buffer. Equal amounts of extracts were loaded into sodium dodecyl sulfate (SDS) polyacrylamide gels for electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% low-fat dry milk for 3 h, and then incubated overnight at 4 °C with primary antibodies against INPP4B, p-SGK3T320, SGK3, p-AKTT308, AKT, p-ERK, ERK (Cell Signaling Technology, MA, USA); p-Ets-1, Ets-1, Flag (Bioworld Technology Inc. MN, USA); NPM1-mA (Abcam, Cambrige, UK) and β-actin (Santa Cruz Biotechnology Inc. CA, USA) as loading control. Membranes were washed in Tris-buffered saline (TBS) (10 mM Tris-HCl pH 8, 150 mM NaCl) containing 0.1% Tween 20, and then incubated with HRP-conjugated secondary antibody for 1 h, and subsequently exposed to enhanced chemiluminescence substrate (Millipore, MA, USA). Membrane blot signals were detected using the Bio-Rad Gel Imaging System on cool image workstation II (Viagene, FL, USA). Quantification of protein expression was normalized against the β-actin protein expression using imaging software.
Jin H., Yang L., Wang L., Yang Z., Zhan Q., Tao Y., Zou Q., Tang Y., Xian J., Zhang S., Jing Y, & Zhang L. (2018). INPP4B promotes cell survival via SGK3 activation in NPM1-mutated leukemia. Journal of Experimental & Clinical Cancer Research : CR, 37, 8.
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2

Anti-FLAG and Phospho-Specific Antibody Protocol

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Anti-FLAG M2 (#F1804) and Anti-FLAG M2-HRP (#A8592) were purchased from Sigma-Aldrich and anti-Src antibody (#MA5-15120) was from Thermo Scientific. p-Src (Y416) (#2113), p-Abl (Y412) (#2865), p-Tyr-100 (#9411), p-Tyr-1000 (#8954), anti-GAPDH (#14C10), Myc Tag (mouse #2276 and rabbit #71D10), p-AKT (T308) (#13038), p-AKT (S473) (#4060), p70 S6 kinase (#9202), and phospho-p70 S6K (T389) (#9234) primary antibodies were purchased from Cell Signaling Technologies (CST). Secondary antibodies Goat anti-Mouse-HRP (#31430) and Goat anti-Rabbit-HRP (#31460) were from Thermo Scientific. Alexa Fluor 488 anti-mouse (#A11029) and Alexa Fluor 647 anti-rabbit (#A21245) were from Life Technologies. All primary antibodies were used at a 1:1000 dilution and secondaries at 1:5000. Protease inhibitor cocktail (#P8340), Phosphatase inhibitor cocktail 2 (#P0044) and 3 (#P5726) were purchased from Sigma-Aldrich. NeuCode labeling reagents L-Lysine:2HCl (3,3,4,4,5,5,6,6-D8, 98%) (#DLM-2641-0) and L-Lysine:2HCl (13C6, 99%, 15N2, 99%) (#CNLM-291-H-0.25) were from Cambridge Isotopes. Rapamycin was from Cayman Chemical and M-PER extraction reagent (#78501) was from Thermo Scientific. All restriction enzymes and DNA polymerases were purchased from NEB (Ipswich, MA). Oligonucleotides and gBlocks Gene Fragments were purchased from IDT and all constructs were verified by DNA sequencing (Quintara Biosciences).
Diaz J.E., Morgan C.W., Minogue C.E., Hebert A.S., Coon J.J, & Wells J.A. (2017). A Split-Abl Kinase for Direct Activation in Cells. Cell chemical biology, 24(10), 1250-1258.e4.
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3

Western Blot Analysis of Signaling Proteins

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Cells were lysed in RIPA buffer complemented with Set I and Set II phosphatase inhibitors at 1× (Calbiochem), and protease inhibitors at 1× (Roche). Whole cell lysate concentration was determined with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad). Proteins were resolved on SDS-PAGE gels and electrotransferred to nitrocellulose membranes, 0.2 µm (Bio-Rad). Primary antibodies pS6S235/236, S6, pAKTS473, AKT, p4E-BP1T37/46, 4E-BP1, Cleaved PARP, pAktT308, p62, Rictor, Raptor, HIF-1α, HIF-2α, pERK1/2T202/Y204 (mouse), ERK, p-p90RSKS380, RSK1/2/3, pBADS112, pBADS136, pEGFRY1068, cleaved-caspase3 were from Cell Signaling Technologies. VHL (Santa Cruz #FL-181). mTOR primary antibody was from Millipore. Primary antibody dilutions were to manufactures' specifications (See Table S1). Tubulin (Sigma #T5168), KU-80 (GeneTex #GTX70485) and Actin-HRP (Santa Cruz #C-11) primary antibodies served as loading controls (LC) where noted. Secondary anti-Rabbit and ant-mouse antibodies were from (Fisher) and diluted in 5% milk, 1× TBS-T solution. ECL Western Blotting Detection reagents (GE Healthcare) were used for developing blots onto autoradiography film. For difficult to detect proteins SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) was used in combination with ECL.
Bailey S.T., Zhou B., Damrauer J.S., Krishnan B., Wilson H.L., Smith A.M., Li M., Yeh J.J, & Kim W.Y. (2014). mTOR Inhibition Induces Compensatory, Therapeutically Targetable MEK Activation in Renal Cell Carcinoma. PLoS ONE, 9(9), e104413.
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4

Nuclear Protein Extraction and Immunoblotting

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Cells were lysed in NP-40 buffer (40 mM HEPES, pH 7.4; 400 mM NaCl; 1 mM EDTA, pH 8.0; 1% NP-40 (CA-630, Sigma); 5% glycerol; 10 mM pyrophosphate; 10 mM β-glycerophosphate; 50 mM NaF; 0.5 mM orthovanadate) containing Protease Inhibitor Cocktail (Sigma) and 1 mM DTT. Nuclear isolation was performed with a Nuclear Extract Kit (40010, Active Motif, Carlsbad, CA, USA), with 10 μg/ml ALLN (208719, Millipore, Bedford, MA, USA) treatment 20 min prior to isolation, and ALLN added to the hypotonic and lysis buffers. The nuclear fraction was washed with hypotonic buffer prior to lysis.
Antibodies used for immunoblots recognized SREBP1 (sc-8984, Santa Cruz, Santa Cruz, CA, USA), SREBP2 precursor and processed C-terminus (557037, BD, Franklin Lakes, NJ, USA), SREBP2 mature N-terminus (30682, Abcam, Cambridge, MA, USA), Actin (A5316, Sigma), and from Cell Signaling Technologies (Danvers, MA, USA): ACC1 (3676), FASN (3180), SCD (2438), HA (2367), P-Akt-T308 (9275), P-Akt-S473 (4051), Total-Akt (4691), P-S6K1-T389 (9234), Total-S6K1 (2708), P-S6-S240/S244 (2215), Total-S6 (2217), 4E-BP1 (9644), Ras (3965), P-Erk1/2-T202/Y204 (9106), Total-Erk1/2 (9102), Lamin A/C (2032), Histone H3 (4499).
Ricoult S.J., Yecies J.L., Ben-Sahra I, & Manning B.D. (2015). Oncogenic PI3K and K-Ras stimulate de novo lipid synthesis through mTORC1 and SREBP. Oncogene, 35(10), 1250-1260.
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5

Immunoblotting of Cell Signaling Proteins

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The following primary antibodies from Cell Signalling Technology (Danvers, MA, USA) were used: 4E‐BP1 (#9452), P‐4E‐BP1S65 (#9451), AKT (#9272), P‐AKTS473 (#9271), P‐AKTT308 (#9275), eIF4E (#2067), mTOR (#2972), P‐mTORS2448 (#2971), raptor (#2280), S6 (#2217), and S6S240/244 (#5364). The primary antibody for α‐actinin (#A7732) was purchased from Abcam (Cambridge, England). The primary antibody for EGFP (11814460001) was from Roche Applied Science (Penzberg, Germany). All Cell Signalling Technology antibodies as well as EGFP were diluted 1:1000; α‐actinin was diluted 1:5000. The secondary antibodies goat anti‐mouse (#115‐035‐003) and goat anti‐rabbit (#111‐035‐003) were purchased from Jackson ImmunoResearch Europe (Ely, Cambridgeshire, England) and were diluted 1:10 000. All antibodies were diluted in 4% bovine serum albumin in TBS‐T.
Ham A.S., Chojnowska K., Tintignac L.A., Lin S., Schmidt A., Ham D.J., Sinnreich M, & Rüegg M.A. (2019). mTORC1 signalling is not essential for the maintenance of muscle mass and function in adult sedentary mice. Journal of Cachexia, Sarcopenia and Muscle, 11(1), 259-273.
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6

Immunoprecipitation and Western Blotting

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Anti-FLAG monoclonal antibody M2 (Sigma-Aldrich), anti-MSI2 monoclonal antibody EP1305Y (Abcam) and normal Rabbit IgG PP64B (Millipore) were used for immunoprecipitation. For Western blotting the following antibodies were used: mouse monoclonal BCAT1 (clone ECA39, BD Transduction Laboratories) and Bcat1 OTI3F5 (OriGene), rabbit monoclonal S6K (#9202 and #2708), pS6K (#9234), AKT (#4691), pAKT, T308 (#13038) and pAKT, S473 (#4060) from Cell Signaling, rabbit monoclonal MSI2 EP1305Y, mouse monoclonal HSP90 F-8 (Santa Cruz Biotech) and mouse monoclonal β-tubulin BT7R (Thermo Fisher Scientific).
Hattori A., Tsunoda M., Konuma T., Kobayashi M., Nagy T., Glushka J., Tayyari F., McSkimming D., Kannan N., Tojo A., Edison A.S, & Ito T. (2017). Cancer progression by reprogrammed BCAA metabolism in myeloid leukemia. Nature, 545(7655), 500-504.
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7

Immunohistochemical Analysis of Prostate Lobes

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Prostate lobes were separated by microdissection, and embedded in paraffin for sectioning 24 (link). After antigen retrieval in hot citrate buffer, sections were blocked in 5% of normal horse serum and 1% of normal goat serum, and subjected to immunohistochemistry staining using the ABC Elite Kit and the DAB Kit (Vector) according to the manufacturers’ recommendations. The following antibodies were used: Ki-67 (ab15580, 1:600) from Abcam; ATF3 (Santa Cruz sc-188, 1:200), AR (sc-816, 1:200), and p63 (sc-8430, 1:200) from Santa Cruz; p-AKT S473 (#4060, 1:200), p-AKT T308 (#2965,1:200), AKT (#4691, 1:200), p-S6 (#2211, 1:400), S6 (#2217, 1:200), cleaved caspase-3 (#9661, 1:300), and Pten (#9188, 1:100)from Cell Signaling; MMP2 (NB200-193, 1:200) from Novus; and MMP-9 (ab137867, 1:1000) from Abcam. For α-smooth muscle actin (α-SMA) staining, sections were incubated with alkaline phosphatase (AP)-conjugated anti-α-smooth muscle actin antibody (Sigma, 1:600) followed by detection of AP activity using the SIGMAFAST Fast Red TR/Naphthol AS-MX tablets (F4523, Sigma) according to the supplier’s protocol. For quantifying IHC staining intensity, random microscopic fields were captured and digitized by a CCD camera (Olympus). Signal intensity was determined using the Image-Pro Plus software and presented as integrated optical density (IOD).
Wang Z., Xu D., Ding H.F., Kim J., Zhang J., Hai T, & Yan C. (2014). Loss of ATF3 Promotes Akt Activation and Prostate Cancer Development in a Pten Knockout Mouse Model. Oncogene, 34(38), 4975-4984.
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8

Immunoblotting Techniques and Antibody Usage

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Immunoblotting was performed as described [6] (link), employing antibodies to phosphotyrosine (4G10, 05-321, EMD Millipore), PTEN (#9552), P-Smad 1/5/8 (#9511), P-Smad 3 (#9520), P-PKC[T514] (#9379), P-PKC[T638/641] (#9375), P-PKC[S660] (#9371), P-S6 (#2211), P-Akt[S473] (#9271), P-Akt[T308] (#9275), P-GSK3[S9] (#9331), and GSK3 (#9315) from Cell Signaling Technology (Danvers, MA) or IGF-1R (sc-713), and E2F4 (sc-866) from Santa Cruz Biotechnology (Santa Cruz, CA). All other antibodies used were purchased from commercial sources and listed previously [11] (link)–[13] (link).
Jahn S.C., Law M.E., Corsino P.E., Davis B.J., Harrison J.K, & Law B.K. (2014). Signaling Mechanisms that Suppress the Cytostatic Actions of Rapamycin. PLoS ONE, 9(6), e99927.
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9

Western Blot Antibody Validation

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Foxo1, pFoxo1-S253, Akt, pAkt-T308, ERK1/2, pERK1/2-T202Y204, GAPDH, and α-actin antibodies were from Cell Signaling Technology (Billerica, MA), and Agt antibody was purchased from Immuno-Biological laboratories, Inc (Japan). Insulin and collagenase were purchased from Sigma, and Percoll from Amersham.
Qi Y., Zhang K., Wu Y., Xu Z., Yong Q., Kumar R., Baker K.M., Zhu Q., Chen S, & Guo S. (2014). Novel Mechanism of Blood Pressure Regulation By Foxo1-Mediated Transcriptional Control of Hepatic Angiotensinogen. Hypertension, 64(5), 1131-1140.
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10

Murine Lung Adenocarcinoma Protein Analysis

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Murine lung adenocarcinoma tissue lysates were prepared and analyzed by immunobloting as previously described [51 (link)]. Briefly, using modified RIPA lysis buffer with protease and phosphatase inhibitors, we lysed the tissues using the tissue lyser (Qiagen) according to manufacturer's protocol. Total proteins were measured using a modified Lowry method. The lysates were denatured via the SDS loading buffer, and SDS-polyacrylamide (4-15%) gel electrophoresis was carried out. Proteins were transferred to nitrocellulose membrane and probed with antibodies against the proteins discussed in the figures. We developed the blots by chemiluminescence using Odessey Fc Imager (Licor). The following antibodies were obtained from Cell Signaling Technology: EGFR; pEGFR(Y1068); AKT; pAKT(T308); ERK; pERK; STAT3; and pSTAT3. Pro-SPC was procured from Millipore and ACTIN from Sigma-Aldrich.
Venugopalan A., Lee M.J., Niu G., Medina-Echeverz J., Tomita Y., Lizak M.J., Cultraro C.M., Simpson R.M., Chen X., Trepel J.B, & Guha U. (2016). EGFR-targeted therapy results in dramatic early lung tumor regression accompanied by imaging response and immune infiltration in EGFR mutant transgenic mouse models. Oncotarget, 7(34), 54137-54156.
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