P akt1

Manufactured by Santa Cruz Biotechnology

Sourced in United States

P-Akt1 is a protein kinase that plays a central role in the PI3K/Akt signaling pathway. It is involved in the regulation of various cellular processes, including metabolism, cell growth, proliferation, and survival.

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Lab products found in correlation

7 protocols using p akt1

Western Blot Analysis of Inflammatory Signaling

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Mouse monoclonal antibodies (STAT3, STAT5, ERK1/2, Akt, NF-κB, IL-6, actin, p-ERK1/2, and p-p38), rabbit polyclonal antibodies (cleaved IL-1β, TLR-2, TLR-4, TLR-5, p38, p-STAT3, p-STAT5, p-Akt1, and p-NF-κB), and goat polyclonal antibodies (TNF-α and IL-1β) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The samples derived from cells and lung homogenates were lysed in radioimmunoprecipitation assay (RIPA) buffer, separated by electrophoresis on SDS-PAGE gels, and transferred to nitrocellulose transfer membranes (GE Amersham Biosciences, Pittsburgh, PA). Proteins were detected by Western blotting using primary Abs at a concentration of 1/200 and were incubated overnight (51 (link)). Labeling of the first Abs was performed using relevant secondary Abs conjugated to horseradish peroxidase (HRP) (Santa Cruz Biotechnology), which were detected using ECL regents (Santa Cruz Biotechnology) and quantified using Quantity One software (Bio-Rad).

Zhang Y., Zhou C.M., Pu Q., Wu Q., Tan S., Shao X., Zhang W., Xie Y., Li R., Yu X.J., Wang R., Zhang L., Wu M, & Deng X. (2019). Pseudomonas aeruginosa Regulatory Protein AnvM Controls Pathogenicity in Anaerobic Environments and Impacts Host Defense. mBio, 10(4), e01362-19.

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Western Blot Analysis of Autophagy Markers

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Mouse monoclonal Abs against LC3, β-Actin, Akt1, and rabbit polyclonal Abs against AnxA2, mTOR, ULK1, p-Akt1, and p-mTOR were obtained from Santa Cruz Biotechnology. Rabbit polyclonal Abs against Fam13A was obtained from Proteintech Group (Chicago, IL), while p-ULK1 (ser317) was obtained from Cell Signaling Technology (Danvers, MA). The samples derived from cells and lung homogenates were lysed in RIPA buffer, separated by electrophoresis on 12% SDS-PAGE gels and transferred to nitrocellulose transfer membranes (GE Amersham Biosciences, Pittsburgh, PA). Proteins were detected by western blotting using primary Abs at a concentration of 1/200 (Santa Cruz Biotechnology) or 1/1000 (Cell Signaling Technology) and were incubated overnight. Labelling of the first Abs was detected using relevant secondary Abs conjugated to HRP (Santa Cruz Biotechnology), detected using ECL regents (Santa Cruz Biotechnology) (38 ). Gel bands were quantified by Quantity One software (Bio Rad) and error bar represents for three independent immunoblotting assays (38 ). Phosphorylated and total protein levels were determined and quantified by three independent successive immunoblotting membranes.

Li R., Tan S., Yu M., Jundt M.C., Zhang S, & Wu M. (2015). Annexin A2 regulates autophagy in Pseudomonas aeruginosa infection through Akt1-mTOR-ULK1/2 signaling pathway. Journal of immunology (Baltimore, Md. : 1950), 195(8), 3901-3911.

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Protein Interaction Detection by GST-Pulldown and IP

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GST pulldown and immunoprecipitation (IP) assays were performed as previously described (Kim et al., 2017 (link)). Briefly, cells were lysed in NP-40 cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, and 5 mM EDTA) and incubated with glutathione-Sepharose 4B beads (GE Healthcare, Piscataway, NJ, United States) or appropriate antibodies for 2 h at 4°C. Immunoblotting using cell lysate or tissue samples was performed as previously described (Cheon and Lee, 2018 (link)). The following antibodies were used to visualize specific proteins: FLAG, MYC, and β-actin (Sigma-Aldrich, St. Louis, MO, United States); GFP, IL-6R, gp130, NEGR1, AKT1, and p-AKT1 (Santa Cruz Biotechnology, Santa Cruz, CA, United States); glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cusabio, College Park, MD, USA); STAT3, p-STAT3 (Cell Signaling Technologies, Beverley, MA, United States), and human Fc (hFc; Gibco, Thermo Fisher Scientific, Waltham, MA, United States).

Yoo A, & Lee S. (2023). Neuronal growth regulator 1 may modulate interleukin-6 signaling in adipocytes. Frontiers in Molecular Biosciences, 10, 1148521.

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BDNF Signaling Pathway Activation

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Recombinant human BDNF, leupeptin, aprotinin, phenyl-methylsulfonyl fluoride, pepstatin A, soybean trypsin inhibitor, sodium fluoride, sodium vanadate, glycerophosphate, 2-mercaptoethanol, NMDA, glycine, Tween 20, NP-40, and Histopaque-1077 were from Sigma. Anti-PSD-95 (05494) was from Millipore. Anti-TrkB (SC-8316), -pTrkB (SC8058), -BDNF/pro-BDNF (SC-2098), -NT3 (SC-547), -NT4 (SC-545), -phosphotyrosine (SC-508), -Erk2 (SC-154, SC-81457), -pErk2 (SC-7383), -pAkt1 (SC-7985-R), -Akt1 (SC-65487), -PLC (SC7290), -NR1 (SC-9058), -NR2A (SC-9056), -actin (SC-376421), -β-actin (SC-47778), and -Arc (SC365736) were from Santa Cruz Biotechnology. Seize-X immunoprecipitation kit, antigen elution buffer, Bind NeutrAvidin, high binding capacity coated 96-well plates, and West Pico chemiluminescent reagents were from Pierce-Endogen. Bradford reagent, SDS-PAGE reagents, and prestained molecular weight markers were from ThermoFisher. Protease inhibitors (EDTA-free) and protein phosphatase inhibitor tablets were from Roche. BDNF was reconstituted according to the manufacturer’s instruction. To avoid freezing damage, 10% glycerol was added to achieve 10 ng/L BDNF and it was stored at −80°C until use. All other test agents were made fresh according to the manufacturer’s recommendation. The DMSO concentration in the incubation medium was 1% when used.

Robinson S., Mogul A.S., Taylor-Yeremeeva E.M., Khan A., Tirabassi A.D, & Wang H.Y. (2021). Stress Diminishes BDNF-stimulated TrkB Signaling, TrkB-NMDA Receptor Linkage and Neuronal Activity in the Rat Brain. Neuroscience, 473, 142-158.

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Western Blot Analysis of Signaling Proteins

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Cells were lysed in NETN lysis buffer containing a protease inhibitor cocktail (Sigma). Equal amounts of proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After blocking, Western blot analysis was carried out using the following primary antibodies: Met (#4560, Cell Signaling), phospho-Met (1234/1235) (#3077, Cell Signaling), Akt1 (sc-8312, Santa Cruz), p-Akt1 (sc-33437, Santa Cruz), Erk1 (sc-93, Santa Cruz), p-Erk1/2 (sc-16982, Santa Cruz), and α-tubulin (T5168, Sigma-Aldrich). IHC staining of phospho-Met was carried out with anti-p-Met (1234/1235) (#3077, Cell Signaling).

Wu C.J., Pan K.F., Chen J.Q., Tao Y.C., Liu Y.C., Chen B.R., Hsu C., Wang M.Y., Sheu B.C., Hsiao M., Hua K.T, & Wei L.H. (2024). Loss of LECT2 promotes ovarian cancer progression by inducing cancer invasiveness and facilitating an immunosuppressive environment. Oncogene, 43(7), 511-523.

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Protein Expression Analysis in BMECs

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Total protein was extracted from the BMECs using RIPA (radio immune-precipitation assay) cell lysis solution (Beyotime, Nanjing, China) after transfection for 72 h. The samples were separated on a 10% SDS-PAGE gel (30 μg of protein per sample) and transferred onto nitrocellulose membranes (Bio-Rad, Shanghai, China). After blocking with 5% skim milk powder solution for 1 h at 37 °C, the membranes were incubated overnight at 4 °C with a murine polyclonal rabbit antibody against CDKN1A, STAT5, p-STAT5, PI3K, AKT1, p-AKT1, mTOR, p-mTOR, and CSN2 (Santa Cruz, CA, USA), and then incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime, Beijing, China). The protein binds were visualized with Super ECL Plus (Solarbio, Beijing, China), and GAPDH (Abcam) was used as an internal reference control. The results reported represent the mean of three independent experiments.

Wu X., Huang J., Liu Y., Li H., Han B, & Sun D. (2022). Bta-miR-106b Regulates Bovine Mammary Epithelial Cell Proliferation, Cell Cycle, and Milk Protein Synthesis by Targeting the CDKN1A Gene. Genes, 13(12), 2308.

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Immunoblotting Antibody Validation Protocol

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The antibody anti-B-actin (#4967) (Cell Singling, USA) was purchased from Cell Signaling (Cell Signaling, MA, USA). The other primary antibodies used in this study were Bax(sc-4780), Bcl2(sc-7382), Bid(sc-6539) HDAC1, (sc-7872), Caspase 3(sc-7148), MMP9 (sc-13520), MMP 2(sc-1359), GAFP(sc-6170), pRB(sc-12901(), Rb(sc-15)N-Myc(sc-42), Id2 (sc-489), VEGF (sc-7269), EGFR2 (sc-271847), P-BAD(sc-133356), pAKT-1(sc-514032) were purchased from Santacruz Biotechnology (Santa Cruz, CA, USA) PI3K (GTX50858) was purchase from Gene Tex, USA, AKT(6744) was purchased Bio vision, USA). Horseradish peroxidase-conjugated secondary anti-rabbit was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-mouse (Santa Cruz, CA, USA)

khathayer F, & Mikael M. (2024). Mocetinostat as a novel selective histone deacetylase (HDAC) inhibitor in the promotion of apoptosis in glioblastoma cell line C6 and T98G. Research Square.

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