The transfected cells were harvested and washed with PBS and then fixed in fresh paraformaldehyde (4%) for 20 min at 37°C. Then, they were treated with Triton X-100 (0.5%) to increase permeability, followed by washing with PBS. After that, the cells were incubated with rabbit or mouse anti-human eNOS (18985-1-AP, Proteintech, USA), p-AKT (66444-1-lg, Proteintech, USA), VEGFA (ab52917, Abcam, Cambridge, MA, USA), or p-VEGFR1 (AF4170, R&D Systems, USA) antibody for 3 h at room temperature. The cells were washed using PBS followed by incubation with goat anti-rabbit/anti-mouse Cy3 or fluorescein isothiocyanate (FITC) antibody for 60 min at room temperature in dark. The cells were then subjected to confocal microscopy for capturing images.
P akt 66444 1 lg
Manufactured by Proteintech
Sourced in United States
P-AKT (66444-1-lg) is a primary antibody product from Proteintech. It is designed to detect phosphorylated AKT (Protein Kinase B), a critical signaling molecule involved in various cellular processes. The core function of this product is to provide a tool for researchers to investigate the activation state of AKT in their experiments.
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Lab products found in correlation
3 protocols using p akt 66444 1 lg
1
Immunofluorescence Staining of Transfected Cells
2
Compound Sourcing and Antibody Validation
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CA was purchased from Sigma-Aldrich (C1129-500G, St Louis, MO, USA). MK-2206 dihydrochloride was obtained from MedChemExpress (HY-108,232, USA). Antibodies against p62 (18420-1-AP), CYP2E1 (19937-1-AP), AKT (10176-2-AP), and p-AKT (66444-1-lg) were from Proteintech Group (Chicago, IL, USA). The mTOR (380,411) antibody was from Zen BioScience (Chengdu, Sichuan, China). p-mTOR (#5536), LC3A/B (#12,741) and Alexa Fluor®594 Conjugate (#8889S) were from Cell Signaling Technology (Danvers, MA, USA). β-actin (AF0003) was purchased from Beyotime Biotechnology (Nanjing, Jiangsu, China). Alexa Fluor 488 (A21206) was from Sigma-Aldrich Co. (St. Louis, MO, USA). Chloroquine (CQ) was form MedChemExpress (HY-17,589 A, USA).
3
Insulin-induced Protein Signaling in HepG2 Cells
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HepG2 cells were inoculated in 6-well plates at 1 × 106 cells/well and divided into control group (no insulin and L-50), model group (insulin pretreatment for 24 h) and treatment group (insulin pretreatment for 24 h followed by different concentrations of L-50). Groups of cells were lysed with RIPA buffer and collected. Primary antibodies include JNK (51151-1-AP, Proteintech, China), phosphorylated JNK (p-JNK, 4668T, CellSignalingTechnology, USA), AKT (10176-2-AP, Proteintech, China), phosphorylated AKT (p-AKT, 66444-1-lg, Proteintech, China), GSK3β (22104-1-AP, Proteintech, China), β-actin (66009-1-Ig, Proteintech, China), and GAPDH (10494-1-AP, Proteintech, China) were used in this study. The dilutions of primary antibodies were as follows: 1:500 for JNK; 1:1000 for p-JNK, GSK3β and GAPDH; and 1:2000 for AKT, p-AKT and β-actin.Protein expression was measured with luminescent solutions, incubated with the relevant secondary antibodies, both of which were at a dilution of 1:2000. Then, the color was developed by a multifunctional molecular imaging system, and the results of relevant protein band densities were analyzed with ImageJ software.
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