The following antibodies were used in this study: LC3 (1:1000) (PM036, MBL International), actin (1:2500) (A2066, Sigma Aldrich), LAMP1 (1:2000) (H4A3, Developmental studies hybridoma bank, developed by August, J.T./Hildreth, J.E.K. obtained under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA). The signaling pathway antibodies were purchased from Cell Signaling Technology unless otherwise stated: phospho-AKT (1:100) (9271), AKT (1:1000) (9272), phospho-MTOR (1:500) (2974), phospho-RPTOR (1:500) (2083), MTOR (1:1000) (2983), RAB5 (1:1000) (3547S), ERM (1:1000) (3142S), cleaved caspase-3 (1:100) (9664), SQSTM1 (1:500) (MABC32, EMD Millipore), DNM1/dynamin-1(1:1000) (SAB450066, Sigma Aldrich), RAB7 (1:1000) (ab50533, Abcam), RILP (1:500) (ab54559, Abcam), ATP6V0A1 (1:500) (ab105937, Abcam), PLIN2/adipophilin (1:500–1:1000) (GP40, Progen). The antibody to CRYBA1 has been described previously (1:1000).12 (link) FITC-phalloidin was obtained from Molecular Probes-Invitrogen (F432). Rapamycin (R8781) and bafilomycin A1 (B1793) were purchased from Sigma.
Phospho akt
Manufactured by Merck Group
Sourced in United States, Germany
Phospho-Akt is a laboratory reagent used to detect and quantify the phosphorylation of the serine/threonine protein kinase Akt. It is a key component of the PI3K/Akt signaling pathway and plays a crucial role in cellular processes such as cell growth, proliferation, and survival. Phospho-Akt is commonly used in research applications to study the activation and regulation of this important signaling molecule.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (4 mentions)
Phospho erk1 2, by Merck Group (3 mentions)
Stat1, by Merck Group (2 mentions)
Alexa fluor 488 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Anti il4 blocking antibody clone 11b11, by Thermo Fisher Scientific (2 mentions)
Anti h3k27ac ab4729 antibody, by Abcam (2 mentions)
Phospo stat1, by Merck Group (2 mentions)
Stat6, by Merck Group (2 mentions)
Phospho stat6, by Merck Group (2 mentions)
Phospho stat1 alexafluor 647, by BD (2 mentions)
Phospho stat6 pe, by BD (2 mentions)
Recombinant mouse ifn γ and il 4, by R&D Systems (2 mentions)
Anti actin a4700, by Merck Group (2 mentions)
Erk1 2, by Merck Group (2 mentions)
Phospho erk1 2, by Cell Signaling Technology (2 mentions)
Fitc phalloidin, by Thermo Fisher Scientific (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
A2066, by Merck Group (1 mentions)
Ab50533, by Abcam (1 mentions)
13 protocols using phospho akt
1
Autophagy Markers and Signaling Pathways
2
Western Blot Analysis of Cardiac Signaling
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Cardiac homogenates were prepared in 300 µl of 10 mM NaHCO3 and 100 µl 20% SDS (sodium dodecyl sulfate). Mixtures were kept at 25°C for 30 min before centrifugation to remove debris. Thereafter, supernatants (called homogenates) were kept at −20°C until further analysis. Western blot analysis was performed as reported (Gergs et al., 2004 (link)). Aliquots of 100 µg of protein were loaded per lane. The antibodies against ERK (extracellular regulated kinase), AKT (protein kinase B), phospho-ERK, phospho-AKT, and A2A-ARs were obtained from Merck (Darmstadt, Germany). All secondary antibodies were conjugated with an alkaline phosphatase (Sigma-Aldrich, Taufkirchen, Germany). Bands were detected using enhanced chemifluorescence (GE Healthcare, Freiburg, Germany), and fluorescent bands were visualized in a Typhoon 9410 PhosphorImager and quantified using the ImageQuaNT software (GE Healthcare, Freiburg, Germany). enhanced chemifluorescence detection was carried out according to the manufacturer’s instructions (GE Healthcare, Freiburg, Germany).
3
Osteoblast Differentiation Molecular Markers
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Unless otherwise specified, all chemicals and reagents were purchased from the Sigma Chemical Company (St. Louis, MO, USA). MGF was purchased from Chemicon International (Temecula, CA, USA). Antibodies to IgG, β-actin, AKT, mammalian target of rapamycin (mTOR), ALP, osteocalcin (OCN), CD44, CD34, MGF receptor, phospho-AKT, phospho-mTOR, and phospho-p90RSK1 (Ser380) were purchased from the Millipore Corporation, USA.
4
Western Blot Antibody Reagents for Signaling Pathways
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Anti-AMPK (#2793) and anti-phospho-AMPK (#2535) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies for detecting phosphatidylinositol-3-kinase (PI3K; #06–497), AKT (#07–416), phospho-AKT (#07–310), p53 (#CBL404), matrix metalloproteinase-2 (MMP-2; #AB19015), and MMP-9 (#AB19016) were from Millipore (Bedford, MA, USA). The antibody for recognizing GAPDH (#NB300-221) was obtained from Novus Biologicals (Littleton, CO, USA). Anti-rabbit (#GTX213110-01) and anti-mouse (#ab6728) secondary antibodies conjugated to horseradish peroxidase (HRP) were respectively purchased from GeneTex (Irvine, CA, USA) and Abcam (Cambridge, MA, USA). ARP101 (#A4433) was obtained from ApexBio Technology (Houston, TX, USA). All chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.
5
Signaling Mechanisms in Macrophage Activation
6
Comprehensive Cell Lysis and Immunoblotting
7
Western Blotting for Protein Analysis
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The cells were harvested 24 h after treatment at the indicated doses and times, and the cell lysates were subjected to western blotting, performed as described previously (20 (link)). Briefly, the cells were collected and lysed using 10 mM Tris, 1 mM ethylenediaminetetraacetic acid, 10 mM KCl and 0.3% Triton X-100 (pH 7.9). The concentration of the protein samples was measured by the Bradford method. The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto Hybond-P polyvinylidene fluoride membranes (Amersham, Piscataway, NJ, USA). The membranes were subjected to western blot analysis with primary antibodies to caspase-8, -9 and -3, poly(ADP-ribose) polymerase (PARP), p110δ, Akt, phospho-Akt, p65, phospho-p65, cyclin-dependent kinase (CDK)4, CDK6, cyclin D1 and β-actin (Sigma-Aldrich). The secondary antibodies used in this study were provided by Multisciences Co., Ltd. (Hangzhou, China).
8
Protein Immunoblotting for Phosphoproteins
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Platelets were lysed with RIPA buffer (Cell Signaling) and then supplemented with a protease inhibitor cocktail (Sigma). Following SDS-PAGE, proteins were transferred to polyvinylidene difluoride membranes and probed with primary antibodies (from Cell Signaling) against phospho-p38, p38, phospho-Akt, Akt, and β-actin (Sigma). Immunoreactivity was detected with ECL (Pierce).
9
Platelet Activation and Signaling Pathways
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ML355, U46619 (Cayman Chemical, MI); CRP-XL (VCPBIO, Shenzhen, China); Thrombin (Roche, Mannheim, Germany); 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), SQ22563, forskolin, and sodium nitroprusside (Sigma-Aldrich, Munich, Germany); phospho-Akt (# 4060), phospho-PI3K (# 17366), phospho-Erk1/2 (# 4370), phospho-p38 (# 9216), phospho-Syk (# 2710), phospho-PLCγ2 (# 3871), caspase-3 (# 9662), and anti-Actin (# 4970) antibodies (Cell Signaling, Frankfurt, Germany); Phospho-Vasodilator-stimulated phosphoprotein (VASP) S239 (Clone 16c2) and phospho-VASPS159 (clone 5C6) (Nano Tools, Teningen, Germany); Fibrinogen-Alexa-Fluor 647, Calcein-AM (Molecular Probes, Göttingen, Germany); Phycoerythrin conjugated CD62P, Phycoerythrin conjugated Annexin-V (BD Bioscience, Heidelberg, Germany); 2`,7`-dichlorodihydrofluorescein diacetate (DCF-DA) (Calbiochem, Schwalbach, Germany), ABT-737 (Selleckchem, Munich, Germany); horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG (Amersham, Freiburg, Germany).
10
Immunoblotting of EGFR and Akt Signaling
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Cells were harvested and lysed in Nonidet P-40 buffer containing a protease-inhibitor mixture (Sigma). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on Mini-PROTEAN TGX Any kD Precast Gels (BioRad, Hercules, CA, USA) and subsequently transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Immunoblots from tumor cell lysates were probed with antibodies against DDX3X (Sigma), EGFR, phospho-EGFR (Tyr1068), phospho-EGFR (Tyr1173), phospho-EGFR (Tyr845), Akt, phospho-Akt, Erk1/2, phospho-Erk1/2, and β-actin (Sigma). All antibodies except for anti-DDX3X and anti-β-actin were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Secondary antibodies consisted of anti-mouse IgG (BioRad) and anti-rabbit IgG conjugated to horseradish peroxidase (Abcam, Cambridge, MA, USA). Immunoreactive protein bands were visualized using an ECL kit (Pierce). At least three independent experiments were performed for all analyses.
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