Western blotting was performed to detect the cellular expression of TRAIL protein in the ADSCs and apoptosis-related proteins (caspase-3, caspase-4, caspase-8 and caspase-9) in A375 cells. A375 cells and ADSCs were co-cultured in a Transwell system for 48 h and then, ADSCs were collected. Equal amounts of whole cell lysates were resolved by 12% SDS-PAGE and electrotransferred onto a polyvinylidene difluoride membrane (Beyotime Institute of Biotechnology). The PVDF membranes were blotted with 5% BSA and then incubated with primary antibodies, including monoclonal mouse anti-human sTRAIL (1:500; #500-M49; PeproTech, Rocky Hill, NJ, USA), polyclonal rabbit anti-human caspase-4 (1:200; #4450S; Cell Signaling Technology, Inc.), caspase-3 (1:500; #AC030), caspase-8 (1:500; #AC056), caspase-9 (1:500; AC062), Akt (1:500; #AA326), phospho-Akt (Ser473; 1:500; #AA329) and β-actin (1:1,000; AA128) antibodies (Beyotime Institute of Biotechnology). After washing 3 times with Tris-buffered saline with Tween 20, the PVDF membranes were incubated with alkaline phosphatase-labeled goat anti-rabbit (1:1,000; #A0239) or anti-mouse (1:1,000; #A0258) IgGs (Beyotime Institute of Biotechnology). The immunoreactive signals were detected using a Gel Docx2000 scanner system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and analyzed with Image J software, version 2.1.4.7 (imagej.nih.gov/ij ).
Phospho akt ser473
Manufactured by Beyotime
Sourced in United States
Phospho-AKT (Ser473) is a laboratory reagent used for the detection and quantification of phosphorylated AKT protein at serine 473 in cellular samples. It functions as a specific antibody that binds to the phosphorylated form of AKT, enabling researchers to analyze the activation status of this important signaling protein.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Beyotime (2 mentions)
β actin, by Merck Group (2 mentions)
Phospho mtor s2448, by Boster Bio (2 mentions)
Goat anti mouse igg h l hrp, by OriGene (2 mentions)
Polyvinylidene difluoride membrane, by Beyotime (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Polyclonal rabbit anti human caspase 4, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bcl 2, by Beyotime (1 mentions)
Odyssey clx infrared fluorescence imaging system, by LI COR (1 mentions)
Cleaved caspase 3, by Beyotime (1 mentions)
Anti phospho trkb, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Roche (1 mentions)
3 methyladenine 3 ma, by Merck Group (1 mentions)
Chloroquine cq, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Gsk3β, by Bioss Antibodies (1 mentions)
Phospho gsk3β, by Bioss Antibodies (1 mentions)
4 protocols using phospho akt ser473
1
Molecular Mechanisms of TRAIL-Induced Apoptosis
2
Protein Extraction and Western Blot Analysis
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Proteins of the neurons were extracted by a RIPA lysis buffer (#P0013C, Beyotime Biotechnology) containing 1% phenylmethylsulfonyl fluoride (PMSF, Roche), followed by centrifuging at 4 °C, 13,000 rpm, 25 min to collect the supernatant. Proteins were denatured and separated by 10% SDS-PAGE, transferred to PVDF (polyvinylidene fluoride) membrane. The primary antibodies were used as follow: Anti BDNF (#AF1423), total-Akt (#AF0045), phospho-Akt (Ser473) (#AF1546), Bcl-2 (#AF0060), β-actin (#AF0003) and cleaved-Caspase-3 (#AF1150) were provided by Beyotime Biotechnology. Anti phospho-TrkB (#4619) antibody was provided by Cell Signaling Technology, Inc. The secondary antibodies including: Goat anti-Mouse IRDye 800CW (#926-32210), Goat anti-Rabbit IRDye 800CW (#926-32211), IRDye 680RD Goat anti-Mouse (#926-68070) and IRDye 680RD Goat anti-Rabbit (#926-68071) were provided by Beijing North Yitao Trading Co., Ltd. Western blot images were scanned by the Odyssey CLx infrared fluorescence imaging system (LI-COR Biosciences). The normalized protein expression level was calculated according to the optical densities of the blots that were quantified by Image J software.
3
Moxifloxacin-Induced Apoptosis and Autophagy
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MOX was purchased from Sigma-Aldrich/Merck KGaA (Darmstadt, Germany; European Pharmacopoeia Reference Standard), and a purity of > 95%. MOX was dissolved in dimethyl sulfoxide (DMSO). FBS was provided by Zhejiang Dayhang biological technology (Hangzhou, China). Penicillin-streptomycin Solution, Annexin V-FITC Apoptosis Detection Kit was purchased from Beyotime Biotechnology (Shanghai, China). MTT, diaminobenzidine (DAB), polyvinylidene fluoride (PVDF) membranes and hematoxylin were purchased from Sigma-Aldrich/Merck KGaA. The reagents 3-methyladenine (3-MA), chloroquine (CQ) and DMSO were obtained from Sigma-Aldrich/Merck KGaA. Antibodies used in this study were against LC3B (CST, 2775S, USA), P62 (CST, 5114T, USA), AKT (CST, 9272S, USA), Phospho-AKT (Ser473) (Beyotime, AA329, China), mTOR (CST, 2972S, USA), Phospho-mTOR (S2448) (Boster, BM4840, China), p70S6K (Bioss, bs-6370R, China), Phospho-p70S6K (Ser417) (Bioss, bs-5668R, China), GSK3β (Bioss, bs-0023RR, China), Phospho-GSK3β (Bioss, bs-0028RR, China), Ki67 (Bioss, bs-23103, China), β-actin (Sigma-Aldrich, A1978, Germany), goat anti-rabbit immunoglobulin G (IgG) (H+L)-horseradish peroxidase (HRP; SunGene GmbH, LK2001, Germany) and goat anti-mouse IgG (H+L)-HRP (OriGene Technologies, ZB-2305, USA).
4
Immunostaining and Western Blotting Protocol
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IVM was purchased from Sigma–Aldrich/Merck KGaA (Darmstadt, Germany; European Pharmacopoeia Reference Standard), purity >95%. IVM was maintained at −20°C or dissolved by dimethyl sulfoxide (DMSO) and stored at −20°C (used in vitro) as stock solutions. Antibodies used in the present study were against LC3B (CST, 2775S, U.S.A.), P62 (CST, 5114T, U.S.A.), AKT (CST, 9272S, U.S.A.), Phospho-AKT (Ser473) (Beyotime, AA329, China), mTOR (CST, 2972S, U.S.A.), Phospho-mTOR (S2448) (Boster, BM4840, China), Ki67 (Bioss, bs-23103, China), β-actin (Sigma–Aldrich, A1978, Germany), goat anti-rabbit immunoglobulin G (IgG) (H+L)-horseradish peroxidase (HRP; SunGene GmbH, LK2001, Germany) and goat anti-mouse IgG (H+L)-HRP (OriGene Technologies, ZB-2305, U.S.A.).
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