Hispolon was acquired from BJYM Pharmaceutical. & Chemical Co., Ltd. (Beijing, China). The purity of hispolon was higher than 95% (Figure 1 A). LPS, dexamethasone (DEX) and other chemicals, solvents, and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits for the determination of cytokine secretion were acquired from BioLegend Inc. (San Diego, CA, USA). Anti-PI3k and Anti-p-AKT were obtained from Merck Millipore (Merck KGaA, Darmstadt, Germany). The antibodies against TLR4, AKT, p-JNK, ATF6, p-CaMKK2, p-LKB1, catalase, GPx, SOD, Keap1, COX-2, caspase 12, IRE1, GRP78, PERK, CHOP, Beclin 1 and LC3 I/II were obtained from GeneTex (Irvine, CA, USA). Antibodies against IKK, p-IKK, JNK, p-ERK, ERK, p-p38, mTOR and p-mTOR were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-iNOS, anti-HO-1, anti-Nrf-2, anti-PPARγ, anti-IκBα, anti-NF-κB, anti-p38, and anti-β-actin were purchased from Abcam (Cambridge, UK). Determination of protein concentration using a Bio-Rad protein assay kit (Bio-Rad Laboratories Ltd., Watford, UK).
Anti pi3k
Manufactured by Merck Group
Sourced in United States
Anti-PI3K is a laboratory reagent that inhibits the activity of the phosphoinositide 3-kinase (PI3K) enzyme. PI3K plays a key role in various cellular processes, including cell growth, proliferation, and survival. Anti-PI3K is a tool used in research to study the effects of PI3K inhibition on cellular function.
Automatically generated - may contain errors
Lab products found in correlation
Anti p akt, by Merck Group (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
P ikk, by Cell Signaling Technology (1 mentions)
P erk, by GeneTex (1 mentions)
Anti pparγ, by Abcam (1 mentions)
Catalase, by GeneTex (1 mentions)
Caspase 12, by GeneTex (1 mentions)
Anti p38, by Abcam (1 mentions)
Anti nrf2, by Abcam (1 mentions)
Anti ho 1, by Abcam (1 mentions)
P jnk, by GeneTex (1 mentions)
P lkb1, by GeneTex (1 mentions)
Keap1, by GeneTex (1 mentions)
Lc3 1 2, by GeneTex (1 mentions)
Anti nf κb, by Abcam (1 mentions)
5 protocols using anti pi3k
1
Hispolon Inhibits Inflammatory Pathways
2
Arterial Tissue Protein Analysis
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Total protein was extracted from arterial tissue (1.0 g) using RIPA lysis buffer (Thermo Fisher Scientific, Inc.). Total protein was quantified using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.) and 40 µg protein/lane was separated using 15% SDS-PAGE. The separated proteins were subsequently transferred onto a nitrocellulose membrane and blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) for 1 h at 37˚C. The membranes were subsequently incubated with the following primary antibodies for 12 h at 4˚C: Anti-PI3K (1:2,000; cat. no. ab232997; Abcam), anti-phosphorylated (p)-PI3K (1:1,000; cat. no. ab182651; Abcam), anti-AKT (1:2,000; cat. no. ab185633; Abcam), anti-p-AKT (1:2,000; cat. no. ab133458; Abcam), anti-mTOR (1:2,000; cat. no. ab32028; Abcam) and anti-β-actin (1:1,000; cat. no. ab8226; Abcam). Following the primary antibody incubation, membranes were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5,000; ab6721; Abcam) for 24 h at 4˚C. Protein bands were visualized using the Pierce™ ECL Western Blotting Substrate (cat. no. 32209; Invitrogen; Thermo Fisher Scientific, Inc.). Protein expression was quantified using Quantity-One version 3.2 software (Bio-Rad Laboratories, Inc.).
3
Comprehensive Protein Expression Analysis
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Western blotting was conducted with the following primary antibodies (all diluted 1:1000): anti-CCR10 (#sc-365957, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GFP (#sc-8334, Santa Cruz Biotechnology), anti-TNF (#3707, Cell Signaling Technology, Danvers, MA, USA), anti-PI3K (#SAB4502195, Sigma-Aldrich, St Louis, MO, USA),anti-Akt (#9272, Cell Signaling Technology), anti-phospho-AktSer473 (p-Akt) (#4060, Cell Signaling Technology), anti-cleaved caspase-3 (#9661, Cell Signaling Technology), anti-cleaved PARP (#9546, Cell Signaling Technology), anti-PCNA (#sc-56, Santa Cruz Biotechnology), and β-actin (#5125, Cell Signaling Technology). Bands were detected with ECL reagents (KeyGen Biotech, Nanjing, China). Band signals were quantified through densitometry. Results were reported as the ratio of the target protein’s densitometry units to β-actin’s densitometry units.
4
Protein Expression Analysis in Cells
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Radioimmunoprecipitation assay lysis buffer was used to extract protein from indicated cells. Bradford protein assay kit (Beyotime, Shanghai, People’s Republic of China) was used to measure the protein concentration. Total 60 μg of protein was separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto 0.22 μm nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk for 2 hours and incubated with primary antibodies (anti-Sall4, anti-PTEN, anti-phosphorylated PTEN, and anti-Bmi-1 [Sigma-Aldrich]; anti-PI3K, anti-pPI3K, anti-AKT, anti-pAKT, anti-Wnt3a, anti-β-catenin, anti-GSK3β, and anti-GSK3β [Cell Signaling Technology, Danvers, MA, USA]; anti-E-cadherin, anti-N-cadherin, anti-fibronectin, anti-vimentin [Abcam, Cambridge, UK]; and anti-GAPDH [Proteintech, Wuhan, People’s Republic of China]) overnight at 4°C. The membranes were washed with tris-buffered saline containing 0.1% Tween 20, and then incubated with appropriate horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, 1:2,000; goat anti-mouse, 1:5,000; Boster, Wuhan, People’s Republic of China) for 1 hour at 37°C. Enhanced chemiluminescence reagent was used to detect the signal on the membrane. The data were analyzed and normalized to the expression of the internal control (glyceraldehyde 3-phosphate dehydrogenase).
5
Immunocytochemistry of Akt Signaling
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Immunocytochemistry was performed and modified according to Iida’s study. Briefly, SH-SY5Y cells were washed with PBS three times, fixed with PBS containing 4% (wt/vol) paraformaldehyde for 15 min, and then permeabilized with 0.5% (wt/vol) Triton X-100 in PBS for 20 min. Immunocytostaining was performed with anti-Akt (1:200), anti-P-Akt (1:200), anti-PI3K (1:200), anti-P-PI3K (1:200), anti-BAD (1:200), anti-Bax (1:100), anti-Bcl-2 (1:100), anti-Cytc (1:200), anti-GSK3β (1:200), anti-p53 (1:100), anti-NGF (1:200), and anti-TrkA (1:200) antibodies (Sigma). After the nonspecific reaction was blocked with PBS containing 10% (wt/vol) bovine serum albumin (BSA), the cells were incubated with the primary antibody in PBS overnight, washed with PBST, and incubated with the second antibody (1:200) in PBST for 1 h. After the samples were washed with PBS three times, they were embedded in DAPI for 5 min and then washed with PBST four times. The images were obtained using an Olympus microscope (Shanghai, China). The mean fluorescence intensity was calculated by Image-Pro software (Meyer, TX, USA).
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