Protein extracts were boiled in RIPA buffer (Beyotime) and separated by sodium dodecyl sulfate–polyacrylamide electrophoresis gel electrophoresis (SDS‐PAGE). The proteins were then transferred to polyvinylidene fluoride membranes (Millipore) and probed with anti‐CD9, anti‐CD63, anti‐ALIX, anti‐TSG101, anti‐EGFR, anti‐FXR1, anti‐TLR‐4, anti‐MMP2, anti‐TGF‐β1, anti‐phospho‐STAT5, anti‐STAT5, anti‐phospho‐ERK1/2, anti‐ERK1/2, anti‐phospho‐AKT, anti‐AKT, anti‐Ki‐67, anti‐PTEN (Abcam), anti‐Bcl‐2, anti‐Bax, anti‐E‐Cadherin, anti‐Vimentin, and anti‐GAPDH antibodies (Cell Signaling Technology). Electrochemiluminescence (Millipore) was applied to determine protein expression levels.
Anti phospho akt
Manufactured by Abcam
Sourced in United States, United Kingdom, Germany
Anti-phospho-AKT is a specific antibody that recognizes the phosphorylated form of the AKT protein. AKT is a serine/threonine protein kinase that plays a crucial role in multiple cellular processes, including cell proliferation, survival, and metabolism. This antibody can be used to detect and quantify the activated, phosphorylated state of AKT in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt, by Abcam (19 mentions)
Anti erk1 2, by Abcam (5 mentions)
Anti phospho pi3k, by Abcam (5 mentions)
Anti pi3k, by Abcam (5 mentions)
Anti akt, by Cell Signaling Technology (4 mentions)
Anti phospho erk1 2, by Abcam (4 mentions)
Anti gapdh, by Abcam (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Anti bcl 2, by Abcam (3 mentions)
Anti gsk3β, by Abcam (3 mentions)
Anti cleaved caspase 3, by Abcam (3 mentions)
Anti phospho gsk 3β, by Abcam (3 mentions)
Anti β actin, by Abcam (3 mentions)
Anti pten, by Abcam (2 mentions)
Anti ki67, by Abcam (2 mentions)
Anti phospho fak, by Abcam (2 mentions)
Enhanced bca protein assay kit, by Beyotime (2 mentions)
Hrp conjugated secondary antibody, by Zhongshan Jinqiao Biotechnology (2 mentions)
Anti phospho stat3, by Abcam (2 mentions)
Anti stat3, by Abcam (2 mentions)
32 protocols using anti phospho akt
1
Comprehensive Protein Expression Profiling of Extracellular Vesicles
2
Western Blot Analysis of TRIM59 and Associated Signaling Proteins
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The RIPA protein lysis buffer (Beyotime, Shanghai, China) was used to split the proteins in tissues or cells, and the protein concentration was measured using a bicinchoninic acid disodium kit (Beyotime). An equivalent amount of cell lysate was separated on a 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes. The membranes were subsequently sealed, and anti-TRIM59 antibody (1: 5000) was added. GAPDH (1: 10 000; Multisciences, Hangzhou, China) as an internal reference. The membranes were incubated with goat anti-rabbit IgG secondary antibody (1: 5000; Bioworld Technology, lnc., St. Louis Park, MN, USA). The intensity of protein bands was analyzed with Image J software (National Institutes of Health, Bethesda, MD, USA). Other primary antibodies used in this study included anti-FAK antibody, anti-phospho-FAK, anti-AKT, anti-phospho-AKT, anti-MMP2 antibody, and anti-MMP9 antibody, which were all purchased from Abcam or Santa Cruz Biotechnology (Dallas TX, USA).
3
Phosphorylated Protein Extraction and Analysis
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The phosphorylated protein extraction was performed using the FOCUS PhosphoRich Kit (G-Bioscience, USA) according to the instructions. Western blotting was performed as previously described. Briefly, equal amounts of protein lysate were separated on 12% SDS gel and then electrotransferred to the PVDF membrane. The membrane was incubated with the different primary antibodies: anti-AKT (Cell Signaling Technology, CST, 1 : 1000), anti-phospho-Akt (Ser473, CST, 1 : 1000), and anti-PAI-2 (Abcam, 1 : 1000 dilution) over night at 4°C. Horseradish peroxidase signals were detected by enhanced chemiluminescence substrate (ECL Plus Western Blotting Detection System, GE Healthcare, Uppsala, Sweden) and captured with a CCD system (image station 2000MM, Kodak, Rochester, NY, USA). The quantitative analysis of these images was performed using Molecular Imaging Software Version 4.0, provided by Kodak 2000MM System [19 (link)]. The optical density was normalized against that of β-actin.
4
Western Blot Analysis of Apoptosis Markers
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Proteins were extracted from cell lysates using RIPA lysis Buffer and the protein concentration was measured using the Enhanced BCA Protein Assay Kit (Beyotime) following to the manufacturer’s recommendations. Proteins were denatured by heat, and then separated by SDS-PAGE electrophoresis, finally transferred to PVDF membranes. The membranes were blocked using 1% bovine serum albumin solution for 1 h at room temperature, and then incubated with primary antibodies, including anti-cytochrome c (1:1000; Abcam), anti-cleaved caspase-9 (1:1000; Abcam), anti-cleaved caspase-3 (1:1000; Abcam), anti-phospho-Akt (1:1000; Abcam), anti-Akt (1:1000; Abcam), anti-phospho-Gsk-3β (1:1000; Abcam), anti-Gsk-3β (1:1000; Abcam), anti-phospho-Stat-3 (1:1000; Abcam), anti-Stat-3 (1:1000; Abcam) anti-COXIV (1:1000; Abcam) and anti-β-actin (1:1000; Zhongshan Jinqiao Biotechnology, Beijing, China) at 4°C overnight, followed by incubation with HRP-conjugated secondary antibody (1:5000; Zhongshan Jinqiao Biotechnology, Beijing, China) at room temperature for 30 min. Protein bands were detected using a BeyoECL Plus Kit (Beyotime) following to the manufacturer’s protocols. Relative densitometry was analyzed using Image J2x analysis software (NIH, U.S.A.).
5
Hippocampal Protein Signaling Pathway Analysis
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Hippocampus samples obtained at the end of the animal experiment were homogenized in lysis buffer containing 1% protease inhibitors. Approximately 15 µg proteins were moved to a new tube and incubated at 100 °C for 5 min for denaturation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was run at 120 V for 45 min. The proteins were transferred to polyvinylidene difluoride membranes and then blocked with 5% non-fat dry milk buffer for 60 min at room temperature (RT). Blots were incubated with anti-phospho-p44/42 mitogen-activated protein kinase (MAPK), anti-p44/42 MAPK (ERK1/2), anti-CREB, anti-phospho-CREB, anti-AKT (Cell Signaling Technology, Boston, MA, USA), and anti-phospho-AKT (Abcam, Hong Kong, China) antibodies overnight at 4 °C. After washing three times, the membranes were incubated with secondary antibody for 45 min at RT. eECL Western Blot Kit (Beijing CoWin Biotechnology, Beijing, China) was used to develop the bands.
6
Quantitative Protein Expression Analysis
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Western blot analysis was performed as previously reported [40 (link)]. The cultured cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, China) supplemented with 1 mM PMSF (Invitrogen), and then, the protein concentration was determined using a BCA protein assay kit (Thermo Fisher). Equal amounts of proteins were separated by 10% SDS-PAGE electrophoresis and transferred to a 0.22-μm PVDF membrane (Millipore Corporation, Billerica, MA, USA). The membrane was blocked with 5% BSA for 1 h in room temperature and was then incubated with the following primary antibodies: anti-myoG (1:1000, Abcam), anti-myoD (1:1000, Abcam), anti-phospho Akt (1:1000, Abcam), anti-Akt (1:1000, Abcam), and anti-β-actin (1:3000, Abcam) at 4 °C overnight. Then, the membrane was incubated with an anti-rabbit (1:5000) or anti-mouse (1:5000) fluorescein-conjugated secondary antibody (Abcam). The immune bands were visualized by Odyssey V3.0 image scanning. All of the procedures were performed three times, and the bands were selected from individual results. Quantitative analysis was performed using ImageJ software; the normalized greyscale was calculated by dividing the individual greyscale of each marker to the β-actin.
7
Western Blot Analysis of Apoptotic Signaling
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Proteins were extracted from cell lysates using RIPA lysis Buffer, and the protein concentration was measured using the Enhanced BCA Protein Assay Kit (Beyotime) following to the manufacturer's recommendations. Proteins were denatured by heat and then separated by SDS‐PAGE electrophoresis, finally transferred to PVDF membranes. The membranes were blocked using 1% bovine serum albumin solution for 1 hour at room temperature and then incubated with primary antibodies, including anti‐cleaved caspase‐3 (1:1000; Abcam), anti‐ROCK1 (1:1000; Abcam), anti‐phospho‐PTEN (1:1000; Abcam), anti‐PTEN (1:1000; Abcam), anti‐phospho‐AKT (1:1000; Abcam), anti‐AKT (1:1000; Abcam), anti‐phospho‐GSK‐3β (1:1000; Abcam), anti‐GSK‐3β (1:1000; Abcam) and anti‐β‐actin (1:1000; Zhongshan Jinqiao Biotechnology) at 4°C overnight, followed by incubation with HRP‐conjugated secondary antibody (1:5000; Zhongshan Jinqiao Biotechnology) at room temperature for 30 minutes. Protein bands were detected using a Immun‐Star HRPKit (Bio‐Rad) following to the manufacturer's protocols. Relative densitometry was analysed using Image J2x analysis software (NIH).
8
Western Blot Analysis of Signaling Pathways
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The cells were lysed in lysis buffer (Beyotime, Shanghai, China) for 15 min on ice, then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After blocking the membrane with 5% low-fat milk for 2 h at room temperature, the membrane was probed with the appropriate primary antibodies for 2 h at room temperature. HRP-conjugated goat anti-mouse and -rabbit IgG (H-L) secondary antibodies were treated with membranes (1:1000; Beyotime, Shanghai, China). The Tanon 5200 chemiluminescence imaging system was utilized to detect bound proteins (Tanon, Shanghai, China). The bands were measured using ImageJ (Version 5.1) and normalized to β-actin levels for quantitative analysis. The primary antibodies used in Western blotting (WB) (anti-β--actin, anti-ERK1/2, anti-phospho-ERK1/2, anti-PI3K, anti-phospho-PI3K, anti-AKT and anti-phospho-AKT) were purchased from Abcam.
9
Evaluation of Signaling Pathways
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Primary antibodies including anti-ERK-1/-2, anti-phospho-ERK-1/-2 (The202/Tyr204), anti-STAT3, anti-phosphor-STAT3 (S727), anti-AKT, anti-phospho-AKT, anti-Ki-67, and secondary antibody conjugated with poly-peroxidase and DAB chromogen for immunohistochemical (IHC) staining were purchased from Abcam (Cambridge, MA, USA) and GeneTex (Hsinchu, Taiwan).
10
Western Blot Analysis of LV Proteins
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Proteins were prepared as described previously.16 (link) Freeze-clamped LV tissues (200–300 mg) were homogenized briefly in 10 volumes of lysis buffer containing (in mM) 20 Tris-HCl (pH, 7.4), 150 NaCl, 2.5 EDTA, 50 NaF, 0.1 Na4P2O7, 1 Na3VO4, 1 PMSF, 1 DTT, 0.02% (v/v) protease cocktail (Sigma-Aldrich), 1% (v/v) Triton X-100, and 10% (v/v) glycerol. The homogenates were centrifuged two times at 20 000 × g at 4 °C for 15 min, and the supernatants were saved as total proteins. Protein concentrations were determined by the BCA method. Equal amounts of proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). Western blot analysis was performed under standard conditions with specific antibodies, including anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-PKCɛ (Ser729), anti-PKCɛ, anti-phospho-GSK-3β (Ser9), anti-GSK-3β, anti-PERK, anti-Xbp-1s, anti-p50-ATF6 (Abcam, London, UK), anti-TRAF2, anti-GRP78, anti-caspase-12 (Abcam), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Antibodies were purchased from Cell Signaling (Danvers, MA, USA), unless noted otherwise. The immunoreaction was visualized using an enhanced chemiluminescent detection kit (Amersham, London, UK), exposed to X-ray film, and quantified by densitometry with a video documentation system (Gel Doc 2000; Bio-Rad).
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