Tumor tissues from the mouse model were fixed in 4% paraformaldehyde and embedded in paraffin. After deparaffinization, the sections were incubated with 10 mM sodium citrate buffer (pH 6.0) and 3% H2O2 to repress the endogenous peroxidase activity, followed by 5% BSA and 0.05% Triton X-100 blocking treatment. For IF assay, primary antibodies used were anti-CD31 (1:20, #ab28364), anti-LYVE-1 (5 μg/ml, #ab14917), anti-bFGF (1:200, #ab8880), and anti-VEGFA (1:250) (Abcam); anti-PDGF (1:200, #GB11261), anti-IL-10 (1:200, #GB11108), and anti-TGF-β (1:200, #GB14152) (Servicebio, Wuhan, China); anti-mTOR (1:200), anti-p-Akt (1:400), anti-p-MAPK (1:200), and anti-CD11b (1:800, #17-0112-8, eBioscience, San Diego, CA, USA); and anti-EGF (1:200, #sc-374255, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cell nuclei were visualized with DAPI (5 ng/ml, Invitrogen, New York, NY, USA) and fluorescence was collected by a fluorescence microscope (Olympus). For HE staining, the sections were stained with HE, and the images were captured by an inverted microscope.
Anti p akt
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-p-AKT is a laboratory reagent used for the detection and quantification of phosphorylated AKT protein in biological samples. AKT is a key signaling molecule involved in cellular processes, and its phosphorylation status is an important indicator of cellular activity and signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt, by Thermo Fisher Scientific (3 mentions)
Anti β actin, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by Thermo Fisher Scientific (2 mentions)
Anti egfr, by Thermo Fisher Scientific (2 mentions)
Fluorescence microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Anti cd11b, by Thermo Fisher Scientific (1 mentions)
Anti il 10, by Wuhan Servicebio Technology (1 mentions)
Anti mtor, by Thermo Fisher Scientific (1 mentions)
Anti lyve 1, by Abcam (1 mentions)
Anti egf, by Santa Cruz Biotechnology (1 mentions)
Anti vegfa, by Abcam (1 mentions)
Anti pdgf, by Abcam (1 mentions)
Anti cd31, by Abcam (1 mentions)
Anti tgfβ, by Thermo Fisher Scientific (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Enhanced chemiluminescence regents, by Bio-Rad (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Goat anti mouse igg hrp, by Abcam (1 mentions)
Evans blue, by Merck Group (1 mentions)
6 protocols using anti p akt
1
Immunohistochemical Analysis of Tumor Angiogenesis
2
Protein Extraction and Western Blot Analysis
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The proteins were extracted as previously described 27 (link),28 (link). The cells in each group were lysed with cell lysis buffer supplemented with PhosSTOP phosphatase inhibitor cocktails (Roche, IN, USA). Thirty minutes later, the cell lysates were centrifuged at 12,000 g at 4 °C for 30 minutes, and then the supernatants were collected. Concentration of protein was performed using the bicinchonininc acid protein assay reagent kit. Same amount of proteins (50 µg) in each group were subjected to 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk for 2 hours, the membranes were incubated with anti-PI3K (eBioscience, CA, USA, 1:1,000), anti-AKT (eBioscience, CA, USA, 1:1,000), anti-p-AKT (eBioscience, CA, USA, 1:1,000), anti-TGF-β (eBioscience, CA, USA, 1:1,000), anti-SMAD7 (eBioscience, CA, USA, 1:1,000) or anti-β-actin (eBioscience, CA, USA, 1:1,000). Twenty-four hours later, the membranes were washed and incubated with secondary antibody, and then detected with the enhanced chemiluminescence regents one hour later (Bio-Rad, CA, USA).
3
Investigating Signaling Pathways in Cellular Processes
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The AKT inhibitor LY294002, GSK3β inhibitor SB216763, NFκB inhibitor Bay 11–7082, FITC-dextran, and Evans blue were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, United States). JNK agonist anisomycin was purchased from Beyotime (Beyotime Biotechnology, Shanghai, China). TNF-α was obtained from R&D Systems (Minneapolis, MN, United States). The following primary antibodies were applied in this study: anti-p-GSK3β, anti-GSK3β, anti-p-JNK, anti-JNK, anti-p-NFκB and anti-NFκB antibodies purchased from Cell Signaling Technology (Danvers, MA, United States); anti-FGF20, anti-VE-cadherin, anti-Claudin-5, and anti-GFAP antibodies obtained from Abcam (Cambridge, MA, United States); and anti-p-AKT, anti-AKT and anti-Occludin antibodies purchased from Invitrogen (Carlsbad, CA, United States) and Santa Cruz Biotechnology (Dallas, TX, United States), respectively. The secondary antibodies used in this study were goat anti-rabbit immunoglobulin G (IgG) H&L (HRP) and goat anti-mouse IgG-HRP purchased from Abcam (Cambridge, MA, United States) and Santa Cruz Biotechnology (Dallas, TX, United States), respectively.
4
Protein Expression Analysis in ESCC
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Proteins from ESCC tissues, paired adjacent normal esophageal epithelial tissues, ESCC cell lines and Het-1A cells were extracted using RIPA buffer (Thermo Scientific, USA), and protein concentrations were detected by BCA Protein Assay kit (Pierce Biotechnology, USA). Protein samples (50 μg) were isolated in 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen, USA) and blocked in 5% non-fat dried milk. Then, the membranes were probed with first primary antibody anti-DESC1 (1:1000, Signalway Antibody, USA), anti-EGFR (1:1000, Invitrogen, USA), anti-p-AKT (1:1000, Invitrogen, USA), anti-AKT (1:1000, Cell Signaling, USA) and anti-GAPDH (1:1000, Invitrogen, USA) and the secondary horseradish peroxidase-conjugated antibody (Invitrogen, USA). GAPDH was used as the internal loading control.
5
Protein Extraction and Western Blot Analysis
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The cells were lysed in RIPA buffer (Sigma-Aldrich). After centrifugation, the protein was extracted, and the concentration was quantified using a BCA assay (Pierce, Rockford, IL, USA). Then, protein samples were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Amersham Pharmacia, Little Chalfont, UK). The primary antibodies used were anti-c-MET (1:1000, Thermo Fisher Scientific), anti-EGFR (1:2500, Invitrogen), anti-t-AKT (1:2000, Cell Signaling), anti-p-AKT (1:500, Invitrogen), and anti-GAPDH (1:1000, Invitrogen), and a secondary horseradish peroxidase (HRP)-conjugated antibody (Invitrogen) was used. GAPDH was chosen as the internal loading control.
6
Western Blot Analysis of Cellular Proteins
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Cells were harvested and then lysed in RIPA buffer with protease inhibitors (Protease Inhibitor Cocktail, Sigma). For the detection of nuclear proteins, the nuclei of ARPE-19 cells were separated by the Nuclei Isolation Kit (Sigma). After the determination of protein concentration, 50 μg protein extracts were resolved in 12% SDS-PAGE and transferred to a PVDF membrane. After blocking by 5% non-fat milk for 1 h at room temperature, membranes were blotted in primary antibodies overnight at 4 °C, followed by the incubation with secondary antibodies conjugated with horseradish peroxidase (1: 3000 dilution; Abcam, Cambridge, MA, USA) for 1 h at 37 °C. The primary antibodies employed for blotting were as follows: anti-bax (1: 1000 dilution; Invitrogen, Carlsbad, CA, USA), anti-bcl-2 (1: 500 dilution; Abcam), anti-p-Akt (1: 1000 dilution; Invitrogen), anti-Akt (1: 1000 dilution; Invitrogen), anti-nuclear factor erythroid 2-related factor (Nrf2; 1: 500 dilution; Abcam), anti-heme oxygenase-1 (HO-1; 1: 500 dilution; Abcam), and anti-β-actin (1: 1000 dilution; Invitrogen). The signals were visualized using an ECL kit (Thermo Fisher Scientific) and Image J software (National Institutes of Health, NIH, Bethesda, MD, USA), respectively.
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