LX-2 cells were rinsed with PBS and immediately solubilized in lysis buffer at 4°C for 30 min. Following microcentrifugation at 14000 g for 5 min, the supernatants were transferred into a new tube, and the sample protein concentrations were determined using the Pierce Protein assay kit (Pierce). The protein mixtures were loaded into each well and separated on 12% SDS-PAGE electrophoresis gels. Following a 2-h run, the proteins were transferred onto nitrocellulose membranes (Amersham Biosciences). The membranes were blocked and subsequently incubated with anti-ETS-1 (Abcam), anti-MMP-1, anti-ERK1/2, anti-phospho-ERK1/2, anti-Akt, anti-phosphor-Akt (R&D Systems) and anti-β-actin (Sigma–Aldrich) antibodies at 4°C overnight. After extensive washing, the membranes were incubated with the secondary antibody for 60 min followed by extensive washes. Specific antibody–antigen complexes were detected with ECL Western blot detection kits (Pierce). All experiments were performed independently three times, and the averages were used for the comparisons. Protein expression was quantified via densitometric analyses of the immunoblots using the Quantity One software.
Anti akt
Manufactured by R&D Systems
Sourced in United States
Anti-Akt is a laboratory reagent that specifically binds and inhibits the Akt protein. Akt is a serine/threonine-specific protein kinase that plays a key role in multiple cellular processes. Anti-Akt can be used to study the function and signaling pathways of Akt in various cell-based and biochemical assays.
Automatically generated - may contain errors
Lab products found in correlation
Anti p akt, by R&D Systems (3 mentions)
Dntps, by Thermo Fisher Scientific (2 mentions)
Anti erk1 2, by R&D Systems (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Anti ets 1, by Abcam (1 mentions)
Anti mmp1, by Abcam (1 mentions)
Anti phospho erk1 2, by R&D Systems (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Bcl 2, by R&D Systems (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh2 da, by Merck Group (1 mentions)
5 5 6 6 tetrachloro 1 1 3 3 tetraethyl imidacarbocyanine iodide jc 1, by Merck Group (1 mentions)
Caspase 3, by R&D Systems (1 mentions)
Anti nrf2, by R&D Systems (1 mentions)
Bcl2 associated x (bax), by R&D Systems (1 mentions)
Interferin sirna transfection reagent, by Polyplus Transfection (1 mentions)
Trypsin edta, by HiMedia (1 mentions)
6 protocols using anti akt
1
Western Blot Analysis of Signaling Proteins
2
Protective Effects of S-Allyl Cysteine
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S-allyl cysteine was purchased from Abcam. Trypan blue, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA),5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), and Wortmannin were purchased from Sigma-Aldrich, USA. Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA solutions were purchased from HiMedia, India. INTERFERin siRNA transfection reagent was purchased from Polyplus, USA. Taq polymerase and dNTPs were purchased from Thermo Fisher Scientific, USA. Random hexamer primer and RiboLock RNase inhibitor, and RevertAid reverse transcriptase were purchased from Thermo Scientific, USA. Anti-α-tubulin antibody was purchased from BioBharati LifeScience Pvt. Ltd., India. Anti-Nrf-2, anti-Akt, anti-p-Akt, Bcl2, Bax, caspase 3, and cytochrome c antibodies were purchased from R&D systems, USA. Primers for real time PCR were purchased from Integrated DNA Technologies, USA.
3
Protein Expression Analysis by Western Blot
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Protein extracts were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore, Germany). Protein expression was detected by western blot analysis. Antibodies used herein including anti-IL-6, anti-TNF-α, anti-p-AKT, anti-AKT, anti-SRC, anti-p-SRC, anti-VEGFA, anti-MAPK1, anti-IL-1β, anti-EGFR and β-actin were obtained from R&D Systems. Band density was quantified using ImageJ software and normalised to the corresponding control group.
4
EGCG Modulates Inflammatory Pathways
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EGCG (purity, 98%) was purchased from Sigma-Aldrich (Merck KGaA). LPS was obtained from Sigma-Aldrich (Merck KGaA). The Cell Titer 96 Aqueous cell viability assay kit was procured from Promega Corporation. ELISA kits for vascular endothelial growth factor (VEGF; cat. no. DY493), Rantes (cat. no. DY478), MCP-1 (cat. no. SMJE00B), ICAM-1 (cat. no. MIC100), matrix metalloproteinase 2 (MMP-2; cat. no. SMMP200) and TNF-α (cat. no. SMTA00B), as well as nitric oxide (NO; cat. no. SKGE001) assay kits were purchased from R&D Systems, Inc. Anti-Akt (cat. no. 4685), anti-nuclear factor-κB (NF-κB) p65 (cat. no. 3034), anti-p38 (cat. no. 9212), anti-Erk (cat. no. 4695), anti-phosphorylated (p)-Akt (cat. no. 4060), anti-p-NF-κB p65 (cat. no. 3033) and anti-p-ERK (cat. no. 4376) were all obtained from Cell Signaling Technology, Inc. Mouse anti-β-actin (cat. no. sc-58673) was obtained from Santa Cruz Biotechnology, Inc.
5
Quantitative Western Blot Analysis
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Aliquots of protein (20 µg) were subjected to western blotting. The membranes were incubated with anti-HIF-1α (BD Biosciences, San Jose, CA, USA), anti-pEGFR (Millipore, Temecula, CA, USA), anti-EGFR (Santa Cruz Biotechnology, Dallas, TX, USA), anti-pErk1/2 (Cell Signaling Technology, Danvers, MA, USA), anti-Erk1/2 (Cell Signaling Technology, Danvers, MA, USA), anti-pAkt (R&D Systems, Minneapolis, MN, USA), and anti-Akt (R&D Systems, Minneapolis, MN, USA) overnight at 4 °C. After washing with TBS/0.1% Tween 20, the membranes were incubated with appropriate secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA). The bands were visualized with Western Blotting Luminol Reagent (Bio Rad, Hercules, CA, USA). Equal loading was verified by reprobing the membranes with an anti-ß-actin (Sigma Aldrich, St. Louis, MO, USA) or anti-GAPDH (BD Biosciences, San Jose, CA, USA) antibody.
6
Protein Expression Analysis in Spinal Cord Injury
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The rats were sacrificed by decapitation at 2 and 8 weeks P.I. (n = 3 per time point per group). The whole brain cortex and a 10-mm lumbar SC segment from each rat were harvested for Western blot analysis. Proteins were separated by 12% SDS-PAGE electrophoresis and were blotted onto a polyvinylidene difluoride membrane. The membrane was subsequently incubated with rabbit polyclonal anti-GFAP (1:200; Thermo Fisher Scientific, Waltham, MA, USA), anti-AKT (1:500; R&D Systems, Minneapolis, MN, USA), anti-Tie2 (1:500; R&D Systems, Minneapolis, MN, USA), anti-AQP4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-AKT and pAKT (1:500; R&D Systems, Minneapolis, MN, USA), anti-Tie2 (1:2000; R&D Systems, Minneapolis, MN, USA), anti-αvβ3 and α5β1 integrins (1:1000; Neuromics, MN, USA), anti-caspase 3 (1:500; Cayman Chemical, Ann Arbor, MI, USA), and mouse anti-MBP (1:1000; Abcam, Cambridge, MA, USA) primary antibodies for 12 h at room temperature. To normalize protein bands to a gel loading control, the membranes were re-probed with rabbit anti-β-actin antibody (1:5,000; Abcam, MA, USA). The membranes were then incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5,000; Santa Cruz, CA, USA) and the blots were detected using enhanced chemiluminescence reagent. The incubation step with the primary antibody was omitted for the negative control.
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