As previously described method (Park et al., 2019 ), the brain hippocampus was homogenized on ice and lysed in a lysis buffer. Protein of 40 μg was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane, which was incubated with mouse β-actin antibody (1:3,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit phosphorylated phosphoinositide 3-kinase (p-PI3K) antibody (1:1,000; Cell Signaling Technology, Danvers, MS, USA), rabbit total PI3K antibody, rabbit phosphorylated protein kinase B (p-AKT) (1:1,000; Cell Signaling Technology), total AKT (t-AKT) (1:1,000; Cell Signaling Technology). Horseradish peroxidase-conjugated anti-mouse for β-actin and anti-rabbit for p-PI3K, t-PI3K, p-AKT, t-AKT were used as secondary antibodies.
Total akt t akt
Manufactured by Cell Signaling Technology
Sourced in United States
Total AKT (t-AKT) is a laboratory equipment product that measures the total amount of AKT protein in a sample, regardless of its activation state. AKT is a key protein involved in cell signaling pathways that regulate cellular processes such as metabolism, proliferation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Phosphorylated akt p akt, by Cell Signaling Technology (3 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Mouse β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Phosphorylated 4ebp1 ser 65, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Phosphorylated akt ser473, by Cell Signaling Technology (1 mentions)
Phosphorylated mtor ser2448, by Cell Signaling Technology (1 mentions)
Anti β actin antibody ab, by Merck Group (1 mentions)
Anti hexokinase 2, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Abcam (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Ecl kit, by PerkinElmer (1 mentions)
Cox 2, by Abcam (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Caveolin 1, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Phosphatase inhibitor, by Solarbio (1 mentions)
Enhanced chemiluminescence, by LI COR (1 mentions)
10 protocols using total akt t akt
1
Hippocampal Protein Analysis
2
Quantitative Western Blot Analysis
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Western blot analysis was conducted according to a previous study [25] (link). Briefly, the equal amounts of proteins obtained from the ileum and jejunum samples were separated by a reducing SDS-PAGE electrophoresis. The proteins were transferred onto PVDF membranes (Millipore, MA, USA) and blocked with 5% non-fat milk in Tris-Tween buffered saline buffer (20 mM Tris, pH 7.5,150 mM NaCl, and 0.1% Tween-20) for 3 hr. The following primary antibodies were incubated overnight at 4°C with gentle rocking: β-actin (Santa Vruz, 1∶400), total 4EBP1 (T-4EBP1, Cell Signaling, 1∶1000), phosphorylated 4EBP1 (Ser 65) (Cell Signaling, 1∶1000), total Akt (T-Akt, Cell Signaling, 1∶1000), phosphorylated Akt (Ser 473) (Cell Signaling, 1∶1000), total mTOR (T-mTOR, Cell Signaling, 1∶1000) or phosphorylated mTOR (Ser 2448) (Cell Signaling, 1∶1000). Then, the HRP-conjugated secondary antibodies were subsequently incubated for 1 hr at room temperature before developing the blots using Alpha Imager 2200 software (Alpha Innotech Corporation, CA, USA). We digitally quantified the resultant signals and normalized the data to the actin abundance. β-actin was used as an internal loading control for cytoplasmic protein fractions.
3
Antibody Selection for Western Blot
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Anti-β-actin antibody (Ab) was purchased from Sigma (St. Louis, MO); anti-Hexokinase II, total Akt (t-Akt), and anti-PDK1 Abs were from Cell Signaling Technology (Beverly, MA); anti-HIF1α Ab was acquired from NOVUS biologicals (Littleton, CO); anti-COX subunit 1 Ab was obtained from MitoSciences (Eugene, OR); anti-phospho-PDH (pPDH), anti-total PDH and CAIX Ab was from AbCam (Boston, MA).
4
Western Blot Analysis of Signaling Proteins
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Protein was extracted from cultured cells with cold lysis buffer containing phosphatase and protease inhibitor cocktail (Sigma). Total proteins were separated by electrophoresis with the 12% SDS-PAGE and then transferred onto nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA). The target proteins were measured using the primary antibodies of COX-2 (Abcam, Cambridge, United Kingdom), phosphorylated AKT (p-AKT, Cell Signaling, Danvers, MA), total AKT (t-AKT, Cell Signaling, Danvers, MA), NFκB-p65 (Abcam, Cambridge, United Kingdom), phosphorylated IκBα (p-IκBα, New England Biolabs, Beverly, MA), total IκBα (t-IκBα, New England Biolabs, Beverly, MA), and followed by detection with the secondary antibody and ECL kit (PerkinElmer, Waltham, MA). β-Actin (Santa Cruz, Dallas, TX) antibodies were used here as an internal control. Quantitative results are expressed as a ratio of target proteins to their internal control and phosphorylated proteins to their total proteins, accordingly.
5
EPC Fractionation and Signaling Pathway Analysis
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After cultivation for 7 days, fractionation of EPCs was performed with RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China), which contained 1% phenylmethanesulfonyl fluoride and phosphatase inhibitor (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The protein concentrations were determined by the bicinchoninic acid method according to the manufacturer’s instructions (Beyotime Institute of Biotechnology, Shanghai, China). Total protein was separated via 10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore) and blocked with 5% skim milk. Primary antibodies against the following proteins were used for western blotting: caveolin-1 (Cell Signaling Technology), total PI3K p85 (t-p-PI3Kp85, Abcam), phosphorylated PI3K p85 (p-PI3Kp85, Abcam), total AKT (t-AKT, Cell Signaling Technology) and phosphorylated AKT (p-AKT, Cell Signaling Technology). The membranes were washed with TBST buffer (Beijing Leagene Biotechnology Co., Ltd.) and probed with HRP-conjugated secondary antibodies (goat anti-rabbit or goat anti-mouse IgG). Protein band and densitometric analyses were performed using enhanced chemiluminescence (LI-COR Biosciences) to quantify the protein concentrations.
6
Investigating TRPM7 Signaling Pathways
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2-APB, propidium iodide (PI) and protease inhibitors cocktail were purchased from Sigma (St. Louis, MO). Lactate dehydrogenase (LDH) assay kit and phosphatase inhibitors cocktail were from Roche (Indianapolis, IN). Fluo-3/acetoxymethyl ester (Fluo-3/AM) was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Mouse monoclonal anti-TRPM7 antibody (Cat# ab85016) and rabbit polyclonal anti-β-actin antibody (Cat# ab8227) were purchased from Abcam (Cambridge, MA). Rabbit polyclonal antibodies against phosphorylated-Akt (p-Akt, Ser473, cat# 4060), p-Akt (Thr308, Cat# 13038), p-ERK1/2 (Thr202/Tyr204, cat# 9101), p-JNK (Thr183/Tyr185 cat# 9251), total-Akt (t-Akt, cat# 9272), t-ERK1/2 (Cat# 9102), t-JNK (Cat# 9258), mouse monoclonal antibody against glial fibrillary acidic protein (GFAP, cat# 3670) and U0126 were purchased from Cell Signaling (Beverly, MA).
7
Extraction and Western Blotting of Proteins
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Total proteins were extracted from cells and tissues using the Pro-Prep lysis buffer (iNtRON Biotechnology, Seoul, Korea), as described previously [37 (link)]. Western blotting was performed using antibodies to JAZF1(Abcam), PPARγ (Santa Cruz), C/EBPβ (Santa Cruz), phospho-AKT (p-AKT; Cell signaling, Beverly, MA), total-AKT (t-AKT; Cell signaling), FABP4 (Santa Cruz), and β-Actin (Santa Cruz) as primary antibodies. Immunoblots were visualized using an ECL detection kit (GE Healthcare, Piscataway, NJ).
8
Emodin-Mediated Neuroprotective Mechanisms
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Emodin (purity ≥ 98%, CAS# 518-82-1) was from Shanghai Base Industry (Shanghai, China). Carboxymethylcellulose sodium (CMC-Na, Cat# 30036365) was provided by Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). EMO was dissolved in 0.5% CMC-Na before use. All sandwich enzyme-linked immunosorbent assay (ELISA) kits such as for IL-1β (Cat# E-EL-R0012c), TNF-α (Cat# E-EL-R2856c) and SOD (Cat# E-BC-K020-M) were from Elabscience Biotechnology (Wuhan, China). Anti-Iba1 (Cat# 019-19741, 1:200) antibody was purchased from Wako (Osaka, Japan). Anti-β-actin (Cat# 20536-1-AP, 1:5000) antibody was obtained from Proteintech (Chicago, USA). Total glycogen synthase kinase 3β (t-GSK3β, Cat# 5676S) and phosphorylated GSK3β (p-GSK3β, Ser9; Cat# 9322S), total Akt (t-Akt, Cat# 9272) and phosphorylated Akt (p-Akt, Ser473; Cat# 4058), and total MAPK ERK1/2 (t-ERK, Cat# 4695) and phosphorylated ERK (p-ERK, Thr202/Tyr204; Cat# 4370) antibodies were purchased from Cell Signaling (Danvers, MA, USA) and diluted 1:1000 for Western blotting. Anti-rabbit (Cat# 926-32210) or anti-mouse IgG (Cat# 926-32211) conjugated to IRDye® 800 CW used in Western blotting, was purchased from Li-Cor Bioscience (Lincoln, NE, USA).
9
Western Blot Analysis of Macrophage Markers
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Cellular proteins were collected using the RIPA lysis buffer (Biouniquer Technology, China), and protein concentration was analyzed on a Nanodrop 2000 spectrophotometry (Thermo Scientific, U.S.A.). Then, total proteins were separated by SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Sigma, U.S.A.). After blocking in 5% skim milk for 2 h, membranes were probed with various primary antibodies including CD163, CD206 (1:500, Santa Cruz, U.S.A.), PTEN, total Akt (tAkt), and phosphorylation Akt (pAkt) (1:1000, Cell Signaling, U.S.A.) overnight at 4° C, followed by incubation with horseradish peroxidase-linked secondary antibodies (Cell Signaling, U.S.A.) for 1 h at room temperature. GAPDH (Sigma, U.S.A.) was used for normalization. Bound proteins were visualized using the ECL Plus Kit (Millipore, U.S.A.) with Image Lab Software (Bio-Rad, U.S.A.).
10
Western Blotting of Insulin Signaling Pathway
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Western blotting was performed, as the previously described method (Kim et al., 2020 (link)). Hippocampal tissues were homogenized on ice and lysed in lysis buffer. The protein content was measured using a colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Thirty micrograms of protein were separated on sodium dodecyl sulfate-polyacrylamide gels and transferred on to a nitrocellulose membrane. Then, incubated with mouse β-actin (1:1,000; Santa Cruz Biotechnology), total IR β (t-IRβ) and phosphorylated IRβ (p-IRβ) (1:1,000; Cell Signaling Technology, Danvers, MS, USA), total IR substrate 1 (t-IRS-1) and phosphorylated IRS-1 (p-IRS-1) (1:1,000; Cell Signaling Technology), t-PI3K and p-PI3K (1:1,000; Cell Signaling Technology), total 3-phosphoinositide dependent protein kinase-1 (t-PDK1) and phosphorylated PDK1 (p-PDK1) (1:1,000; Cell Signaling Technology), total-Akt (t-Akt) and phosphorylated Akt (p-Akt) (1:1,000; Cell Signaling Technology), total glycogen synthase kinase 3 beta (t-GSK3β) and phosphorylated GSK3β (p-GSK3β) (1:1,000; Cell Signaling Technology). Horseradish peroxidase-conjugated secondary anti-mouse antibodies were used for β-actin and horseradish peroxidase-conjugated secondary anti-rabbit antibodies were used for t-IRβ, p-IRβ, t-IRS-1, p-IRS-1, t-PDK, p-PDK, t-PI3K, p-PI3K, t-Akt, p-Akt, t-GSK3β, and p-GSK3β.
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