Phosphor akt

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

Phosphor-AKT is a laboratory equipment product designed to detect and quantify phosphorylated AKT, a key protein involved in cell signaling pathways. It is intended for use in research applications.

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Lab products found in correlation

75 protocols using phosphor akt

Western Blot Analysis of EMT and Stemness Markers

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The ADSCs and MDA-MB-231 cells collected from the co-culture system or GDF15-treated MDA-MB-231 cells were lysed with RIPA lysis buffer. Protein concentrations determined using Bio-Rad protein assay (BIO-RAD, Hercules, CA, USA). Equal amounts of total proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane (IBFP0785C, Merck Millipore, Burlington, MA, USA). After blocking with 5% non-fat milk, the membrane was incubated with primary antibodies at 4 °C overnight. Primary antibodies used in western blotting included β-actin (GTX109639, GeneTex, Hsinchu, Taiwan), α-tubulin (GTX112141), ZEB1 (GTX105278), Snail (GTX82509), ZO-1 (GTX108627), Nanog (GTX100863), Oct4 (GTX101497), β-catenin (ab16051, Abcam, Cambridge, MA, USA), GDF15 (PAB31426, Abnova, Taipei, Taiwan), phosphor-AKT (4060, Cell Signaling, Danvers, MA, USA), and AKT (4691, Cell Signaling). Immunoreactive proteins were detected after incubation with horseradish peroxidase-conjugated secondary antibody (31430, Thermo Scientific) for 1 h at room temperature. The immunoblots were visualized using chemiluminescence reagent (WBKLS0500, Merck Millipore) and quantified by ChemiDoc XRS+ imaging system (BIO-RAD).

Huang J.Y., Wang Y.Y., Lo S., Tseng L.M., Chen D.R., Wu Y.C., Hou M.F, & Yuan S.S. (2019). Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer. Cancers, 12(1), 29.

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Signaling Pathways in Neutrophil Activation

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Phorbol ester phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-Phe (fMLF), C5a and isoluminol were purchased from Sigma-Aldrich (St. Louis, MO, USA). The p38 MAPK inhibitor SB203580 was ordered from Selleck (Houston, TX, USA). Antibodies including Phosphor-Akt, Akt, Phosphor-p38 MAPK, p38 MAPK, MK2, and GAPDH were ordered from Cell Signaling Technology (Danvers, MA, USA). Anti-p47^phox antibody was purchased from Santa Cruz Biotechnology and anti-phospho-p47^phox (Ser329) antibody was generated by N.J. Compass Biotechnology (Nanjing, China). Other reagents were ordered from Sigma-Aldrich (St. Louis, MO, USA).

Sun L., Wu Q., Nie Y., Cheng N., Wang R., Wang G., Zhang D., He H., Ye R.D, & Qian F. (2018). A Role for MK2 in Enhancing Neutrophil-Derived ROS Production and Aggravating Liver Ischemia/Reperfusion Injury. Frontiers in Immunology, 9, 2610.

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Integrin-mediated Signaling Pathway Analysis

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Antibodies: Anti-phospho-ERK (#4370), -phosphop38 (#4831), phosphor-AKT (#4058), and—phospho-Smad1/5 (#9511) were from Cell Signaling (Ozyme, St Quentin en Yvelines France). Anti-β1 integrin (MB1.2) was from BD Biosciences (Le Pont de Claix, France). For IHC, β1 integrin clone 4B7R antibody (Abcam, Paris, France) was used. The anti-phosphotyrosine (PY) monoclonal antibody 4G10 used as hybridoma supernatant was produced in our laboratory. Anti-Actin (clone AC-40), anti -Type I Collagen and doxycycline were from Sigma-Aldrich (L’Isle d’Abeau, France). BMP2 was from Shenandoah Biotechnology Inc. and was used at a 200μg/mL concentration. pEl-BRE4xLuc plasmid was a gift from J. Massagué (NY, USA).

Brunner M., Mandier N., Gautier T., Chevalier G., Ribba A.S., Guardiola P., Block M.R, & Bouvard D. (2018). β1 integrins mediate the BMP2 dependent transcriptional control of osteoblast differentiation and osteogenesis. PLoS ONE, 13(4), e0196021.

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Biochemical Assay Reagents and Antibodies

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Trypsin and bafilomycin A1 were purchased from Sigma Chemical Co., (St. Louis, MO, USA). Anti-GAPDH monoclonal antibody from Calbiochem-Merck Chemicals Ltd. (Nottingham, UK). MnSOD, catalase and OGG-1 antibodies from Adipogen (San Diego, CA, USA). Phospho-mTOR, mTOR, phosphor-AKT, AKT, LC3, p62, LAMP-I, Cathepsin B, HexA and galectin-3 were obtained from Cell Signaling Technology. A cocktail of protease inhibitors (complete cocktail) was purchased from Boehringer Mannheim (Indianapolis, IN, USA). Grace’s insect medium was purchased from Gibco. The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA, USA).

Castejón-Vega B., Rubio A., Pérez-Pulido A.J., Quiles J.L., Lane J.D., Fernández-Domínguez B., Cachón-González M.B., Martín-Ruiz C., Sanz A., Cox T.M., Alcocer-Gómez E, & Cordero M.D. (2021). L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation. Cells, 10(11), 3122.

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Metabolic Reprogramming in Lung Cancer

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The normal human bronchial epithelioid cell (HBE) and the non-small-cell lung cancer cell lines including NCI-H1650, NCI-H1299, A549, NCI-H460, HCC4006, NCI-H1975, and NCI-H358 were obtained from ATCC and cultured with recommended culture medium. The primary antibodies for western blotting including anti-TRAF6 (#8028), hexokinase-1 (#2024), hexokinase-2 (#2867), GLUT1 (#12939), PKM2 (#4053), LDHA (#35 82), VDAC-1 (#4661), phosphor-Akt (#4060), Akt (#8596), phosphor-S6 (#4858), and ubiquitin (#58395) as well as the secondary anti-rabbit IgG HRP (#7074) were products of Cell Signaling Technology Inc. (Danvers, MA). β-Actin (A5316) was obtained from Sigma-Aldrich. In immunohistochemistry staining, the primary antibodies against hexokinase-2 (ab227198) and Ki67 (ab15580) were products of Abcam. TRAF6 shRNA#1 (TRCN0000007350) and shRNA#2 (TRCN0000007351) were purchased from the Sigma Mission shRNA library. The constitutively active Akt (CA-Akt) plasmid (Cat. #10841) and pLKO.1 GFP shRNA (Cat. #30323) were purchased from Addgene (Cambridge, MA, USA). Recombinant human insulin-like growth factor 1 (IGF-1) was a product of R&D (Cat. 291-G1-200). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA).

Feng L., Feng S., Nie Z., Deng Y., Xuan Y., Chen X., Lu Y., Liang L, & Chen Y. (2021). TRAF6 Promoted Tumor Glycolysis in Non-Small-Cell Lung Cancer by Activating the Akt-HIFα Pathway. BioMed Research International, 2021, 3431245.

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Western Blot Analysis of Signaling Proteins

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Cells were lysed in HNTG buffer [20 mM HEPES pH7.4, 150 mM NaCl, 10 % glycerol, 1 % Triton X-100, 1.5 mM MgCl2, 1.0 mM EGTA and proteinase inhibitor cocktail (Roche)]. Cell lysates were subjected to SDS-PAGE separation. Immunoblotting was performed with antibodies against Spry4, EGFR, ERK, β1-integrin, β3-integrin and Src (Santa Cruz), phosphor-ERK, phosphor-Akt, Akt, pSrc (Cell Signaling), and tubulin (Sigma).

Jing H., Liaw L., Friesel R., Vary C., Hua S, & Yang X. (2016). Suppression of Spry4 enhances cancer stem cell properties of human MDA-MB-231 breast carcinoma cells. Cancer Cell International, 16, 19.

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Immunoblotting Analysis of Signaling Pathways

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Cells were lysed in RIPA lysis and extraction buffer (Thermo Fisher Scientific, Cat.#89900) containing 1xprotease inhibitors (Roche, Cat.#11-836-145-001) and phosphatase inhibitor (50 μM NaF and 100 μM Na₃VO₄). Immunoblotting was performed as described(33 (link)) using antibodies that specifically recognize MET (Cell Signaling Technology, Cat.#8198), Phospho-MET (Cell Signaling Technology, Cat.#3077), STING (Cell Signaling Technology, Cat.#13647), CD73 (Cell Signaling Technology, Cat.#13160), TBK1 (Cell Signaling Technology, Cat.#3013), Phospho-TBK1 (Cell Signaling Technology, Cat.#5483), IRF3 (Cell Signaling Technology, Cat.#11904), phospho-IRF3 (Cell Signaling Technology, Cat.#4947), STAT1 (Cell Signaling Technology, Cat.#9172), phosphor-STAT1 (Cell Signaling Technology, Cat.#9167), ERK(Cell Signaling Technology, Cat.#9107), phosphor-ERK (Cell Signaling Technology, Cat.#4370), AKT(Cell Signaling Technology, Cat.#9272), phosphor-AKT(Cell Signaling Technology, Cat.#4060), FRA1 (Abcam, Cat.#124722), phospho-FRA1 (Cell Signaling Technology, Cat.#5841), cGAS(Cell Signaling Technology, Cat.#15102), and β-actin (Cell Signaling Technology, Cat.#3700). Secondary antibodies were from LICOR Bioscience: IRDye 680LT Goat anti-Mouse IgG (#926–68020), IRDye 800CW Goat anti-Rabbit IgG (#926–32211). Imaging of blots are was performed using LICOR Odyssey system.

Yoshida R., Saigi M., Tani T., Springer B.F., Shibata H., Kitajima S., Mahadevan N.R., Campisi M., Kim W., Kobayashi Y., Thai T.C., Haratani K., Yamamoto Y., Sundararaman S.K., Knelson E.H., Vajdi A., Canadas I., Uppaluri R., Paweletz C.P., Miret J.J., Lizotte P.H., Gokhale P.C., Jänne P.A, & Barbie D.A. (2022). MET-Induced CD73 Restrains STING-Mediated Immunogenicity of EGFR-Mutant Lung Cancer. Cancer Research, 82(21), 4079-4092.

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Protein Expression Analysis of Myoblasts

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Myoblasts were harvested and lysed in RIPA buffer supplemented with protease inhibitors (Complete, Roche) and 0.2 nM PMSF. Proteins (20 μg) were denatured and subjected to 10% SDS/PAGE electrophoresis. After transfer to PVDF membrane, blots were treated according to standard procedures. Probing of the blots was performed using phosphor-Akt (1:1000, Cell Signaling Technology, Beverly, MA, USA), anti-androgen receptor (AR, 1:1000, DSHB, Iowa City, IA, USA), PAX7 (1:1000, DSHB, Iowa City, IA, USA) and GAPDH antibody (1:5000, Novus Biologicals, Littleton, CO, USA) as primary antibodies. After 1 h incubation with anti-mouse or anti-rabbit peroxidase-conjugated second antibody (1:5000, Santa Cruz, Dallas, TX, USA), blots were developed by enhanced chemiluminescence (GE Healthcare, Chicago, IL, USA).

Li D., Wang Q., Shi K., Lu Y., Yu D., Shi X., Du W, & Yu M. (2020). Testosterone Promotes the Proliferation of Chicken Embryonic Myoblasts Via Androgen Receptor Mediated PI3K/Akt Signaling Pathway. International Journal of Molecular Sciences, 21(3), 1152.

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Comprehensive Western Blot Analysis of Cellular Proteins

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The Western blot analysis was performed as described in a previous study [27 ]. The blotting bands were then incubated with primary antibodies overnight at 4 °C. The antibodies against the following proteins were used: Caspase-3 (Cell Signaling Technology #14220, 1:1000), PARP (Cell Signaling Technology #9542, 1:1000), BCL2 (Cell Signaling Technology #15071, 1:1000), PROCA1 (Biorbyt #orb1703, 1:1000), ZFP36L2 (Santa Cruz #sc-365908, 1:1000), c-Myc (Cell Signaling Technology #9402, 1:1000), phosphor-Akt (Cell Signaling Technology #4060, 1:1000), Akt (Cell Signaling Technology #4691, 1:1000), phosphor-PI3K (Cell Signaling Technology #17366, 1:1000), and PI3K (Cell Signaling Technology #4292, 1:1000). The internal reference antibodies were as follows: GAPDH (Cell Signaling Technology #5174, 1:1000) and β-tubulin (Proteintech #10068-1-AP, 1:1000). The secondary antibodies were as follows: HRP-conjugated Affinipure goat anti-rabbit immunoglobulin G (IgG) (H + L) (Proteintech #SA00001-2, 1:5000), and HRP-conjugated Affinipure goat anti-mouse IgG (H + L) (Proteintech #SA00001-1, 1:5000) were purchased from Proteintech (IL, USA). The images were captured by chemiluminescence imaging (Bio-Rad, CA, USA) and quantitated using the Quantity One system (Bio-Rad).

Tao X., Li Y., Fan S., Wu L., Xin J., Su Y., Xian X., Huang Y., Huang R., Fang W, & Liu Z. (2023). Downregulation of Linc00173 increases BCL2 mRNA stability via the miR-1275/PROCA1/ZFP36L2 axis and induces acquired cisplatin resistance of lung adenocarcinoma. Journal of Experimental & Clinical Cancer Research : CR, 42, 12.

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Protein Expression Analysis by Western Blotting

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Protein expression was determined by Western blotting, which was performed according to the method of Lee et al. [49 (link)]. Briefly, proteins from cellular lysates were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore, Danvers, MA, USA). After blocking for 1 h with 5% skim milk in TBST buffer, the membranes were incubated overnight at 4 °C with antibodies specific for the following proteins: iNOS (BD Biosciences, San Jose, CA, USA), COX-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD80, CD86, TNF-α, phosphor-PI3K p85 (Tyr458), PI3K, phosphor-AKT (T308), AKT, phospho-p70S6K (T389), p70S6K, phosphor-NF-κB (Ser536), NF-κB, PDK1, LDHA (Cell Signaling Technology; Danvers, MA, USA), HIF1-α (Novus Biologicals, Building IV Centennial, CO, USA), and β-actin (Sigma). After three washes in TBST, membranes were incubated with HRP-conjugated secondary antibodies (GeneTex, Irvine, CA, USA). HRP was detected using the ECL reagent (BioNote, Hwaseong, Gyeonggi-do, Korea).

Kim Y.J., Lee S., Jin J., Woo H., Choi Y.K, & Park K.G. (2022). Cassiaside C Inhibits M1 Polarization of Macrophages by Downregulating Glycolysis. International Journal of Molecular Sciences, 23(3), 1696.

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