Western blot analysis was performed as previously described [14 (link)]. The following commercial antibodies were used in this study: Gab2 (OriGene Technologies, USA), E-cadherin, vimentin, MMP7 and MMP9 (Abcam, UK), phospho-AKT, total AKT, phospho-ERK1/2 and total ERK1/2 (Invitrogen, USA), GAPDH and β-actin (Immunology Consultants Laboratory, USA).
Phospho akt
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Phospho-Akt is a laboratory reagent used to detect and quantify the phosphorylated form of the Akt (Protein Kinase B) protein. Akt is a key regulator of cellular processes such as metabolism, cell growth, and survival. The phosphorylated form of Akt is an important marker for the activation of the PI3K/Akt signaling pathway, which is involved in various biological and pathological processes. Phospho-Akt can be used in a variety of analytical techniques, including Western blotting, ELISA, and immunohistochemistry, to study the activation of the Akt signaling pathway in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Total akt, by Thermo Fisher Scientific (2 mentions)
Total erk1 2, by Thermo Fisher Scientific (2 mentions)
Phospho erk1 2, by Thermo Fisher Scientific (2 mentions)
Phospho mtor, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Mmp 9, by Abcam (1 mentions)
Vimentin, by Abcam (1 mentions)
E cadherin, by Abcam (1 mentions)
Phospho nf κb p65, by Abcam (1 mentions)
Total nf κb p65, by Abcam (1 mentions)
Phospho iκbα, by Abcam (1 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
Vegfr2, by Abcam (1 mentions)
Phospho pi3k, by Cell Signaling Technology (1 mentions)
Stat3, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Cytiva (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
P44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions)
7 protocols using phospho akt
1
Comprehensive Protein Expression Analysis
2
Western Blot Analysis of Signaling Proteins
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Western blot analysis was performed as previous described [19 (link)]. The following commercial antibodies were used in this study: VEGF, EGFR, phospho-c-Myc, total c-Myc, phospho-NF-κB/p65, total NF-κB/p65, phospho-IκBα and total IκBα (Abcam, UK), phospho-AKT, total AKT, phospho-STAT3, total STAT3, phospho-ERK1/2 and total ERK1/2 (Invitrogen, USA), GAPDH and β-actin (Immunology Consultants Laboratory, USA).
3
Western Blot Analysis of Signaling Pathways
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The procedure of Western blot analysis was descried as previously [10 (link), 13 (link)]. In brief, the equal amounts (50 μg/lane) were extracted from the calf muscle tissue or HUVEC and separated by SDS-PAGE using 10~15% acrylamide gradients. The membranes were incubated with antibodies against Phosphoinositide 3-kinases (PI3K), Phospho-PI3K (1:1000; Cell Signaling, Danvers, MA, USA), AKT, Phospho-AKT (1:1000, Invitrogen, Thermo Fisher Scientific, MA, USA), STAT3 (1:1000; Abcam, Cambridge, MA, USA), Phospho-STAT3(Tyr705) (1:1000; Cell Signaling, Danvers, MA, USA), Matrix metallopeptidase 9 (MMP9) (1:1000, Arigo Biolaboratories, Taiwan), vascular endothelial growth factor (VEGF) (1:1000, Proteintech, Rosemont, IL, USA), VEGFR2 (1:1000) and Phospho-VEGFR2 (1:5000 Abcam, Cambridge, MA, USA). Signals were detected with HRP-conjugated goat anti-mouse or goat anti-rabbit IgG (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA). Immunoreactive bands were detected by enhanced chemiluminescence (ECL; Thermo Fisher Scientific, CA, USA) and then were exposed to ECL-Western blotting system (AVEGENE CHEMX 400). The intensity of the protein band was quantified by Image J software (Bethesda, NIH, MD, USA) and the results are expressed as normalized ratio to housekeeping gene GAPDH.
4
Comprehensive Immunoblotting of Cell Signaling
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Whole cell protein lysates were prepared using cell lysis buffer (Cell Signaling Technologies), 1 mM phenylmethylsulfonyl fluoride, proteasome (Roche) and a phosphatase inhibitor cocktail (Sigma-Aldrich). Proteins were resolved on 4-15% gradient gels (Biorad), transferred onto polyvinylidene fluoride membranes (Amersham), and subjected to immunoblotting using primary antibodies from Cell Signaling Technologies: p44/42 MAPK (ERK1/2; #9102), phospho-ERK1/2 (phospho-p44/42 MAPK Thr202/Tyr204; #4376), AKT (#9272), phospho-AKT (Ser473; #4060), TSC1 (#6935), phospho-TSC2 (Ser664; #40729), phospho- TSC2 (Tyr1571; #3614), TSC2 (#4308), phospho-mTOR (Ser2481; #2974), phospho-mTOR (Ser2448; #2971), mTOR (#2983), NF1 (#14623), MEK (#9122S), phosphor-MEK (#9154), vinculin (#4650); from ThermoFisher Scientific: GAPDH (#AM4300); from Millipore: anti-pan_Ras (clone RAS 10 MABS195) from Sigma: LZTR1 (HPA071248). Corresponding horseradish perodixase-conjugated secondary antibodies (Promega) were used for chemiluminescent detection.
5
Honokiol and Rapamycin Signaling Pathways
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Honokiol (HNK) and Rapamycin (RAPA) were purchased from Selleckchem. For Western blot analysis, primary antibodies against CDK2, CDK4, CDK6, Cyclin D1, p21, Bcl-2, Bcl-xL, c-Met, phospho-c-Met, HO-1, Akt, mTOR and S6 and species-specific secondary antibodies were obtained from Cell Signaling. Antibody against β-actin was purchased from Sigma Aldrich. Anti-PD-L1, phospho-Akt, phospho-S6 and phospho-mTOR antibodies were purchased from Thermo Fisher Scientific. Recombinant human hepatocyte growth factor (HGF) was purchased from Peprotech.
6
Protein Extraction and Western Blot Analysis
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Adipocytes were lysed with cell lysis buffer (10 mmol/L Na2HPO4, 50 mmol/L β-glycerophosphate disodium salt hydrate, 50 mmol/L NaF, 1 mmol/L EDTA, 1 mmol/L EGTA, pH 7.4) supplemented with protease inhibitors and 0.5 mmol/L dithiothreitol, homogenized by shearing through a 22G needle, and cleared by centrifugation at 17,000×g for 10 min at 4 °C. Tissues were homogenized by Potter-Elvehjem tissue grinder in Tissue Protein Extraction Reagent (Thermo) supplemented with protease inhibitors and 0.5 mmol/L dithiothreitol and cleared by centrifugation at 17,000×g for 10 min at 4 °C. Protein concentrations were determined using BCA assay (Pierce). Western blots were probed as indicated with β-actin (#A228, Sigma), α-tubulin (T9026, Sigma), GAPDH (#5174), TBP (#8515), A1R (#PA1-041a, ThermoFisher), phospho-Akt (S473, #9271), phospho-MAPK (T202/Y204, #9101), phospho-GSK3α/β (S21/S9, #9331), phospho-P70 S6K (T389, #9205), phospho-GYS (S641, #3891), total Akt (#2920), total P42/P44 MAPK (#4696), total GSK3β (#9315), total GYS (#3893), and FOXO1 (#2880).
7
Cardiac Tissue Protein Analysis
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A portion of cardiac tissue was homogenized as described by Ye et al. [17 (link)]. Protein samples (50 μg for Akt, Phospho-Akt and 90 μg for eNOS) were run with 4–20% Tris–HCl ready gel (Thermo Scientific) or 6% SDS-PAGE, respectively and transferred to pure nitrocellulose membrane. The membranes were incubated overnight at 4 °C with primary antibodies against eNOS (1/1000, Becton Dickinson (DB) Transduction Laboratories, USA), Akt (1/1000, Cell Signaling Technology, Inc.), Phospho-Akt (Ser473)(1/1000, Cell Signaling Technology, Inc.) and β-actin (1/5000, Sigma) and secondarily with HRP-conjugated anti-mouse or anti-rabbit antibodies (Santa Cruz Biotechnology, Inc.). The immunoblots were developed using ECLTM Western Blotting Detection Reagent (AmershamTM). The protein signals were quantified using the MicroChemi 4.2 system (DNR Bio-Imaging Systems Ltd., Israel). The intensity of each protein signal was normalized to the corresponding β-actin stain signal. Data are expressed as ratios between the protein and the corresponding β-actin signal density.
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