Proteins from vascular tissue and cultured VSMCs were lysed by protein lysis buffer. Proteins were run on 8–10% SDS-PAGE to separation and then electro-transferred to PVDF membranes (Millipore). The membranes were blocked with 5% milk in TTBS for 2 h at room temperature and incubated overnight at 4 °C using the primary following antibodies: anti-IL-1β (1:1000, Proteintech, 16,806–1-AP), anti-IL-6 (1:1000, Proteintech, 21,865–1-AP), anti-TNF-α (1:1000, Proteintech, 6029–1-Ig), anti-PTEN (1:1000, Abcam, ab32199), anti-pan-AKT (1:1000, Abcam, ab8805), anti-pan-AKT (phospho T308; 1:1000, Abcam, ab38449), and 1:1000 anti-β-actin (1:2000, Santa, sc-47778). In the next day, after washing in the TTBS for three times, membranes were incubated with a 1:5000 dilution of anti-rabbit or anti-mouse antibody (Santa Cruz) for 1 h at room temperature. Protein bands were detected by enhanced chemiluminescence (ECL) Fuazon Fx (Vilber Lourmat).
Anti pan akt
Manufactured by Abcam
Sourced in United States, China, United Kingdom
Anti-pan AKT is a lab equipment product that can be used to detect the presence and quantify the levels of AKT proteins in biological samples. It is a primary antibody that binds to all isoforms of the AKT protein, allowing for the identification and analysis of total AKT expression.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ab8805, by Abcam (2 mentions)
Anti e cadherin, by Abcam (2 mentions)
Anti n cadherin, by Abcam (2 mentions)
Anti β actin antibody, by Merck Group (2 mentions)
Anti ndfip2, by Bioss Antibodies (2 mentions)
Anti il 1β, by Proteintech (1 mentions)
Anti il 6, by Proteintech (1 mentions)
Fuazon fx, by Vilber (1 mentions)
Ab32199, by Abcam (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Anti n cadherin, by Proteintech (1 mentions)
Anti p akt, by Abcam (1 mentions)
Anti vimentin, by Proteintech (1 mentions)
Anti snail, by Proteintech (1 mentions)
Anti α sma, by Proteintech (1 mentions)
Anti p pi3k, by Abcam (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Anti pi3k, by Abcam (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
6 protocols using anti pan akt
1
Western Blot Analysis of Vascular Protein Markers
2
Evaluating Epithelial-Mesenchymal Transition and PI3K/AKT Signaling
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For the sake of detecting the protein expression of CLCA4 epithelial-mesenchymal transition markers and key elements of PI3K/AKT signaling, western blot was carried out. Cells were lysed and centrifuged. The supernatant was collected and denatured. Proteins were separated SDS-PAGE and blotted onto PVDF membrane. The membrane was then treated with TBST containing 50 g/L skimmed milk at room temperature for 4 hours, followed by incubation with the primary antibodies: anti-CLCA4 anti-E-cadherin (1: 1000, Proteintech), anti-N-cadherin (1: 1000, Proteintech), anti-Vimentin (1: 2000, Proteintech), anti-α-SMA (1: 500, Proteintech), anti-Snail (1: 200, Proteintech), anti-PI3K (1: 2000, Abcam), anti-p-PI3K (1: 1000, Abcam), anti-pan AKT (1: 500, Abcam), anti-p-AKT (1: 5000, Abcam), and anti-GAPDH (1: 1000, Proteintech) respectively, at 37°C for 1 hour. Membranes were rinsed and incubated for 1 hour with the correspondent peroxidase-conjugated secondary antibodies. Chemiluminescent detection was performed with the ECL kit (Pierce Chemical, Rockford, IL, USA). The amount of the protein of interest was normalized to the densitometric units of GAPDH.
3
Protein Expression Profiling in Diabetic Mouse Atria
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Protein was extracted from mouse atrial tissue (Con n=5, DM n=5) after sufficient grinding and lysis. The antibodies used in the experiment were as follows: anti-PARP-1 (1:3000, ab227244; Abcam, Cambridge, UK), anti-AMPK (1:3000, ab32047; Abcam, Cambridge, UK), anti-Sirt1 (1:1000, cst9475; Cell Signaling Technology Inc., Danvers, MA, USA), anti-pan-Akt (1:500, ab8805; Abcam, Cambridge, UK), anti-IKKα (1:10,000, ab32041; Abcam, Cambridge, UK), anti-NF-κB (1:1000, cst8242; Cell Signaling Technology Inc., Danvers, MA, USA), anti-NLRP3 (1:3000, ab214185; Abcam, Cambridge, UK), and anti-GAPDH (1:5000, sc-25778; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).
4
Culturing Human Hepatoma and Kidney Cells
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Human hepatoma Huh7 and Huh7.5.1 cells and human embryonic kidney HEK 293T cells were cultured in Dulbecco modified eagle medium (Life Technologies, United States) supplemented with 10% fetal bovine serum, 1 × non-essential amino acids, 100 IU/mL streptomycin and penicillin, and 2 mM L -glutamine (Gibco, Invitrogen). Cells were incubated under an atmosphere with 5% CO2 at 37°C for subsequent use in experimental procedures. Perifosine and SC79 were purchased from Selleck Chemicals (Houston, TX) and dissolved in dimethyl sulfoxide to generate 10 mM storage solutions for further dilution. Anti-HCV core, anti-SRB1, anti-CLDN1, anti-GAPDH antibodies, anti–pan-AKT, anti-pAKT(T308), and anti-pAKT(S473) primary antibodies were obtained from Abcam, whereas anti-CD81 and anti-OCLN antibodies and Alexa 488– and horseradish peroxidase–conjugated anti–rabbit and anti–mouse immunoglobulin G secondary antibodies were obtained from Invitrogen, with all antibodies used at appropriate dilution ratio according to the manufacturer’s instructions.
5
Western Blotting Protocol for Protein Analysis
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Western blotting was performed as previously described [25 (link)]. The primary antibodies used for Western blotting were as follows: anti-NDFIP2(1:1000, Bioss Antibodies Inc., Beijing, China; Cat # bs-19059R), anti-pan AKT (1:1000, Abcam, Cambridge, UK; Cat # ab8805), anti- phospho-AKT1 (1:1000, Abcam, Cat # ab66138), anti-N-cadherin (1:1000, Abcam, Cat # ab18203), anti-E-cadherin (1:10,000, Abcam, Cat # ab40772). An anti-β-actin antibody (1:2000, Sigma, USA) was used as an internal control to ensure equal amounts of protein loading.
6
Western Blot Analysis of Protein Markers
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The cells were washed with PBS, lysed with radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) containing 1% 10 µM Phenylmethanesulfonyl uoride (PMSF) and 1% phosphatase inhibitor. Quantitative protein concentration using bicinchoninic acid kit (BCA, Vazyme Biotechnology Co., Ltd., Nanjing, China). After protein reduction and denaturation, the equal amount of protein lysis and Page Ruler prestained protein ladder were loaded into 10% SDS-PAGE gel, then transferred to polyvinylidene uoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5 % bovine serum albumin (BSA) and incubated (overnight, 4°C) with primary antibodies. Western blot was performed using antibodies that anti-NDFIP2(1:1000, Bioss antibodies Inc., Beijing, China. Cat # bs-19059R), anti-pan AKT (1:1000, Abcam, Cambridge, UK, Cat # ab8805), anti-AKT1 phospho (1:1000, Abcam, Cat # ab66138), anti-N cadherin (1:1000, Abcam, Cat # ab18203), anti-E cadherin (1:10000, Abcam, Cat # ab40772). After washings with TBS-T, membranes were incubated with the secondary antibody for 1 hour at room temperature. Protein bands are visualized with Western chemiluminescent HRP substrate (Millipore, Billerica, MA, USA). Anti-β-actin antibody (1:2000, Sigma, USA) is used as an internal control to ensure the equal amount of protein loading.
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