Protein from unstained paraffin-embedded samples was deparaffinized and rehydrated via successive washes in xylene/ethanol and water. After incubation in extraction buffer at 100 °C for 20 min followed by a 2 h incubation at 80 °C, proteins were obtained by centrifugation. Whole cell lysates were prepared with RIPA lysis buffer containing protease and phosphatase inhibitor cocktail. Protein concentrations of cell lysates were determined with the bicinchoninic acid protein assay. A total of 30-40 μg of lysate were loaded onto a 10% SDS-PAGE gel and subjected to gel electrophoresis. Resolved proteins were transferred to a polyvinylidine fluoride membrane. Membranes were then blocked in 5% non-fat dry milk in Tris-buffered saline with Tween (TBS-T) for 1 h and then incubated with antibodies to GAPDH, CaMKIIα (Cell Signaling Technology), ERK1/2, phospho-ERK1/2, p38, p-p38, MMP2, MMP9 and TIMP-1 at 4 °C overnight. Membranes were washed with TBS-T and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The blotting bands were visualized with enhanced chemiluminescence. GAPDH was used as a loading control.
Camkiiα
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
CaMKIIα (Calcium/calmodulin-dependent protein kinase II alpha) is a protein that plays a crucial role in calcium signaling and synaptic plasticity. It is a member of the CaMKII family of serine/threonine-specific protein kinases. CaMKIIα is the predominant isoform found in the brain and is essential for various neuronal functions, including learning and memory formation.
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Lab products found in correlation
Gapdh, by Cell Signaling Technology (2 mentions)
Anti mouse igg, by Cell Signaling Technology (1 mentions)
Hrp linked anti rabbit igg, by Cell Signaling Technology (1 mentions)
β tubulin, by Abcam (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Parvalbumin, by Merck Group (1 mentions)
Coxiv, by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (1 mentions)
C fos, by Abcam (1 mentions)
Actin antibody, by Merck Group (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Bdnf antibody, by Santa Cruz Biotechnology (1 mentions)
Pcreb, by Merck Group (1 mentions)
Glua1, by Merck Group (1 mentions)
Glua2, by Merck Group (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Psd95, by Cell Signaling Technology (1 mentions)
Phospho camkiiα, by Cell Signaling Technology (1 mentions)
Phospho pka, by Cell Signaling Technology (1 mentions)
6 protocols using camkiiα
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Signaling Proteins
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The membranes were blocked using 5% BSA in TBST (#ST023; Beyotime, Shanghai, China) at room temperature for 1 h. Western blot assays were performed using antibodies directed against phosphorylated ERK 1/2, ERK 1/2, phosphorylated p65, p65, CAMKIIα (1: 1000; Cell Signaling Technology, United States), and β-Tubulin (1: 5,000; Abcam, UK). Correspondingly, β-Tubulin was used as a loading control. The membrane was incubated with primary antibodies at 4 °C overnight, followed by incubation with HRP-linked anti-rabbit IgG (#7074; Cell Signaling Technology) or anti-mouse IgG (#7076; Cell Signaling Technology) at room temperature for 1 h. The protein bands were visualized using a Pierce ECL Western blotting substrate (#32106; Thermo Fisher Scientific, United States). Images were acquired using a ChemiDoc XRS+ System (Bio-Rad, United States).
3
Immunohistochemical Analysis of Mouse Brain Tissue
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Brain tissue samples (n = 5 mice/goup) were fixed in 10% formaldehyde and embedded in paraffin. Subsequently, 4-μm sections were either stained with hematoxylin for morphological examination or used for immunohistochemistry analysis. The following antibodies were used for immunohistochemistry: p-S6 (#4858, Cell Signaling Technology, Tokyo, Japan) at 1:100, c-FOS (ab208942, Abcam, Cambridge, UK) at 1:200, Parvalbumin (SAB4200545, Sigma) at 1:100, CaMKII-α (#11945, Cell Signaling Technology) at 1:100, COXIV (#459600, Invitrogen) at 1:200, GFAP (#12389, Cell Signaling Technology) at 1:100, and MAP2 (ab5392, Abcam) at 1:2000. The stained proteins were visualized using a Biozero confocal microscope (Keyence Co., Osaka, Japan).
4
Biochemical Characterization of Synaptic Proteins
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All chemicals were purchased from Sigma (St Louis, USA), unless stated otherwise. Antibodies specific for pAKT, AKT, pCaMKIIα, CaMKIIα, pERK1/2, and ERK1/2 were from Cell Signaling Technology (Beverly, USA); brain-derived neurotrophic factor (BDNF) antibody was from Santa Cruz (Dallas, USA); PSD-95 antibody was from Thermo Scientific Pierce; GluA1, pCREB, GluA2, and pSer831 GluA1 antibodies were from Millipore (Darmstadt, Germany); pSer845 GluA1 antibody was from PhosphoSolutions (Aurora, USA); and actin antibody was from Sigma. The purity of AA3 was >95% according to nuclear magnetic resonance analysis.
5
Protein Signaling Pathway Evaluation
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Rabbit primary antibodies used were as follows: K48 polyubiquitin (1:1000; Cell Signaling Technology 8081), phospho-Rpt6 (Ser120; 1:1000; Signalway Antibody 12880), Rpt6 (1:1000; Cell Signaling Technology 13392), phospho-PKA (Thr197; 1:1000; Cell Signaling Technology 5661), PKA (1:1000; Cell Signaling Technology 4782), phospho-CaMKIIα (Thr286; 1:1000; Cell Signaling Technology 12716), CaMKIIα (1:1000; Cell Signaling Technology 4436), PSD95 (1:1000; Cell Signaling Technology 2507), Arc/Arg3.1 (1:1000; Cell Signaling Technology 65650), and GAPDH (1:1000; Cell Signaling Technology 2118).
6
Protein Expression Analysis of Hepatic Cells
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The P62 (Cell Signaling Technology, Danvers, Massachusetts, USA), LC3A/B (Cell Signaling Technology, Danvers, Massachusetts, USA), Alb (Santa Cruz Biotechnology, Inc, Dallas, Texas, USA), Aldob (Santa Cruz Biotechnology, Inc, Dallas, Texas, USA), Wnt3a (Abcam, Cambridge, UK), β-catenin (Abcam, Cambridge, UK), Sox9 (Merck KGaA, Darmstadt, Germany), Wnt5a (Abcam, Cambridge, UK), CaMKIIα (Cell Signaling Technology, Danvers, Massachusetts, USA), Phospho-CaMKIIα (Thr286) (Cell Signaling Technology, Danvers, Massachusetts, USA), and Actin (Bioworld Technology, Bloomington, USA) antibodies were used for Western blotting staining. HGF (315–23) and EGF (400–25) were obtained from Peprotech. OSM (495-MO) was purchased from R&D Systems. ITS-X (51500–056) was obtained from Lifetech. 2-Mercaptoethanol (21985023) was purchased from Thermo Fisher Scientific. VitC (A4403) and nicotinamide (N0636) were purchased from Sigma-Aldrich. Dexamethasone and gentamicin were purchased from Mengchao Hepatobiliary Hospital of Fujian Medical University.
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