Antibodies against CaMKII, pCaMKII, AMPK, pAMPK, mTOR, pmTOR, S6K, pS6K, 4E-BP, ACC, pACC, and Cleaved caspase-3 were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against NQO were purchased from Invitrogen (Carlsbad, CA, USA). Anti-β-actin, anti-rabbit IgG, and anti-mouse IgG antibodies were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
Camkii
Manufactured by Cell Signaling Technology
Sourced in United States, China
CaMKII is a protein kinase that is activated by calcium/calmodulin. It plays a key role in various cellular processes, including synaptic plasticity, gene transcription, and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
P camkii, by Cell Signaling Technology (13 mentions)
Gapdh, by Cell Signaling Technology (6 mentions)
β actin, by Cell Signaling Technology (5 mentions)
Pvdf membrane, by Merck Group (4 mentions)
P ampk, by Cell Signaling Technology (2 mentions)
P mtor, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
C myc, by Abcam (2 mentions)
P creb, by Cell Signaling Technology (2 mentions)
Gsk3β, by Cell Signaling Technology (2 mentions)
β catenin, by Abcam (2 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions)
Phospho camkii thr286, by Cell Signaling Technology (2 mentions)
β actin, by Abcam (2 mentions)
P camkii thr286, by Cell Signaling Technology (2 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
P parkin, by Cell Signaling Technology (2 mentions)
Phospho camkii, by Cell Signaling Technology (2 mentions)
32 protocols using camkii
1
Antibody Immunoblotting Panel
2
Western Blot Analysis of Protein Signaling
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Protein lysates were prepared in RIPA lysis buffer, and separated on 10% SDS–polyacrylamide gel electrophoresis gels by electrophoresis. Subsequently, the lysates were transferred to nitrocellulose membranes and probed with the following antibodies and concentrations: HIF-2α 1:1,000 (Novus #NB100-122), HIF-1α 1:1,000 (Cayman Chemicals #1006421), caspase-3 1:1,000 (Cell Signaling #9662 Danvers, MA, USA), GAPDH 1:2,000 (Cell Signaling #2118), β-tubulin 1:1,500 (Cell Signaling #2146), phospho-4E-BP1 1:1,000 (S65, Cell Signaling #9451), 4E-BP1 1:1,000 (Cell Signaling #9452), phospho-S6K1 1:1,000 (T389, Cell Signaling #9205), c-Myc 1:5,000 (Abcam #32072), S6K1 1:1,000 (Cell Signaling #2708), phospho-AKT 1:1,000 (S473, Cell Signaling #9271), AKT 1:1,000 (Cell Signaling #9272), phospho-EGFR 1:1,000 (Y1068, Cell Signaling #3777), EGFR 1:1,000 (Cell Signaling #4267) ANO1 1:500 (Abcam ab64085), phospho-CAMKIIα 1:1,000 (T286, Cell Signaling #12716) and CAMKII 1:1,000 (Cell Signaling #11945). Uncropped immunoblot images are included in Supplementary Fig. 8 .
3
Hippocampal Protein Expression Analysis
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Protein was extracted from mice's hippocampal tissue. Protein lysates were separated by 12% SDS-PAGE electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 3% BSA for 1 h, the membranes were incubated with primary antibodies. CAMKII (Cell Signaling Technology, 7546, 1 : 500), P-CaMK-II (Cell Signaling Technology, 9546, 1 : 500), P-CREB (Cell Signaling Technology, 9198 s, 1 : 500), CREB (Cell Signaling Technology, 9197, 1 : 500), PKA (Proteintech, 55388-1-AP, 1 : 1000), NMDAR1 (Cell Signaling Technology, 5104 s, 1 : 1000), BDNF (Cell Signaling Technology, 47808, 1 : 1000), PSD95 (Cell Signaling Technology, #2507, 1 : 1000), GAPDH (Proteintech, 6004-1-lg, 1 : 2000) and Tubulin (Proteintech, 10094-1-AP, 1 : 2000) were used at 4°C overnight. The next day, blots were washed 4 times in TBST, followed by incubation with secondary antibodies for 1 h. After washing for 3 times, the blots were visualized using the Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.). BDNF and pro-BDNF were normalized to Tubulin bands, and P-CERB and total CREB bands were taken as a ratio of Tubulin bands. All experiments were performed 3 times.
4
Western Blot Analysis of CaMKII Protein
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The samples were homogenized in 0.1% SDS buffer (Roche, USA) and the lysate was centrifuged at 12,000 rpm for 15 min. The supernatant was collected and the protein concentration was determined using a BCA protein assay kit (Bio-Rad, USA). The extracted protein was separated on SDS-PAGE gel and transferred onto PVDF membrane (Millipore, USA). The membrane was blocked with 5% bovine serum albumin for 1 h to reduce non-specific binding. Then, the blot was incubated with the primary antibody for 12 h at 4°C. The antibodies (CaMKII and β-actin) were purchased from Cell Signaling Technology and used at manufacturer-recommended dilutions (1:1000). After washing, the blot was incubated with HRP-conjugated secondary antibody (Santa Cruz, USA) for 1 h at room temperature. Finally, the signal was detected using the enhanced chemiluminescence kit (Amersham Biosciences) and exposed to X-film.
5
Modulation of Calcium Signaling Pathways
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N,N,N′,N′ -Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) and thapsigargin (TG) were obtained from Sigma (St. Louis, MO, United States). BAPTA-AM, EGTA-AM, H89, 2APB, and stattic were purchased from MCE (NJ, United States). KN-93 and KN-92 were obtained from Selleck (Houston, TX, United States). Antibodies including anti-p-STAT3, -STAT3, -p-CaMKII, -CaMKII, -GAPDH, and the secondary antibody were obtained from Cell Signaling Technology (Beverly, MA, United States). Anti-IP3R,-p-RyR2 and -SERCA2 were purchased from Abcam (Cambridge, United Kingdom). Anti-RyR2 was purchased from Proteintech Group (Chicago, IL, United States). Anti-ZIP9 was obtained from Biorbyt (Cambridge, United Kingdom). Fluorescence dyes were obtained from Invitrogen (Carlsbad, CA, United States).
6
Evaluating Odonto/Osteogenic Protein Expression
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For evaluating the expression of odonto/osteognic proteins, nDPSCs and iDPSCs treated or untreated with baicalin or BMS345541 or DKK1 were harvested, washed and lysed. To analyze the expressions of NF-κB and β-catenin/Wnt pathway related proteins, the cytoplasm protein as well as nucleoprotein were extracted from iDPSCs treated with 20 µM baicalin for 0, 15, 30, and 60 min. After loading the protein onto a 10% SDS-PAGE gel for electrophoresis, electroblotted (Bio-Rad, USA) the protein onto 0.22 μm polyvinylidene fluoride (PVDF) membrane (Millipore, USA) at 300 mA for 1 h. After blocking membranes with solution at 25℃, incubated them with primary antibodies (DSP, Santa Cruz; RUNX2, Abcam; OSX, Abcam; OCN, Abcam; phosphor-P65; Cell Signaling, Boston, MA, USA, P65, Cell Signaling, Boston, MA, USA; phosphor IκBα, Cell Signaling; IκBα, Cell Signaling; β-catenin, Abcam; GSK-3β, Cell Signaling; NLK, Cell Signaling; CaMKII, Cell Signaling; β-ACTIN, Bioworld, Minneapolis, MN, USA; Histone, Bioworld) at 4 °C all night long. Membranes were then washed for 1 h with PBST blended with secondary antibodies (Boster, China) at 37 °C before visualized with ImageQuant LAS4000 system (GE Healthcare, USA). β-ACTIN and H3 were set as the internal parameters, respectively.
7
Pharmacological Neuromodulation Assay
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Remifentanil was purchased from the Yichang Renfu Pharmaceutical Industry. Esketamine was bought from Jiangsu Hengrui Medicine. N-Methyl-d -aspartic acid (NMDA) was purchased from Selleckchem. Polyvinylidene fluoride (PVDF) membrane was bought from Millipore. Antibodies used in this study were purchased from Abcam (CaMKII and p-CaMKII) and Cell Signaling Technology (GluN2B and GAPDH). All other chemicals were purchased from Sigma-Aldrich.
8
Western Blot Analysis of LV Proteins
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Total protein extracted from homogenized LV tissues was electrophoresed in 12% polyacrylamide gel and transferred to PVDF membrane (Millipore, Billerica, MA, USA). The blotted membranes were incubated with antibodies against Akt (1:1,000, catalog #9272, Cell Signaling Technology, Danvers, MA, USA), p-Akt (1:1,000, catalog #9271, Cell Signaling Technology, Danvers, MA, USA), β-arrestin-2 (1:1,000; catalogue #3857, Cell Signaling Technology, Danvers, MA, USA), CaMKII (1:1,000, catalogue #3362, Cell Signaling Technology, Danvers, MA, USA), p-CaMKII (1:1,000, catalog #12716, Cell Signaling Technology, Danvers, MA, USA). After three washes, the blotted membranes were then incubated with horseradish peroxidase-conjugated rabbit secondary antibody (1:5,000, catalog 31460, Thermo Scientific, Waltham, MA, USA). The antigen-antibody complexes were detected with ECL chemiluminescence reagents (catalog RPN2106, GE Healthcare, Piscataway, NJ, USA) and the densitometry was visualized using a LAS-3000 imaging system (FUJIFILM, Kanagawa, Japan).
9
Western Blot Analysis of Protein Expression
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Total proteins from the tissues and cell lines were extracted in lysis buffer (Thermo Fisher Scientific, Rockford, IL, USA) and quantified using the Bradford method. Equal amounts of extracted protein (60 μg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred onto a polyvinylidene fluoride membrane. The membrane was incubated overnight at 4 °C with the following antibodies-TRPV3 (1:100, Abcam, Cambridge, MA, USA), CaMK-II (1:1000), p-CaMK-II (1:1000), CyclinA (1:1000), CyclinD1 (1:1000), CyclinE (1:1000), CDK2 (1:1000), CDK4 (1:1000), P27 (1:1000) (Cell Signaling Technology, Boston, MA, USA), β-actin(1:2000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with peroxidase-coupled anti-mouse/rabbit IgG at 37 °C for 2 h, the protein bands were visualized using ECL (Thermo Fisher Scientific) and detected using the BioImaging Systems (UVP Inc., Upland, CA, USA). The relative amounts of protein were calculated with reference to the amount of β-actin protein.
10
Western Blot Analysis of Signaling Proteins
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Cultured cells and whole tissues extracts were prepared with RIPA buffer supplemented with protease inhibitor cocktail (MERCK P8340), and phosphatase inhibitor cocktails (MERCK P5726 and P0044). Western blotting was performed using the Mini-PROTEAN Tetra Cell electrophoresis system, and transferred to PVDF membranes. The following primary antibodies concentrations were used p-CaMKII 1:1000 (Abcam ab182647), CaMKII 1:1000 (Cell Signaling 3362), GAPDH 1:10,000 (Abcam ab181602), p-ERK1/2 1:20,000 (MERCK M9692), ERK1/2 1:40,000 (MERCK M5670), p-RXR 1:1000 (Affinity Biosciences), RXR antibody 1:200 (SCBT sc-553), p-RYR 1:2000(Abcam ab59225), RYR 1:1000 (ab2868), p-Rac1 1:1000 (Millipore 07-896-I) Rac1 1:1000 (Millipore 05-389), (and Vinculin (provided by Benny Geiger, Weizmann Institute of Science). Horseradish peroxidase conjugated secondary anti-mouse, anti-rabbit or anti-goat was used to detect proteins (Jackson Immunology). Western blots were imaged using the Chemidoc Multiplex system (Bio-rad) and Image Lab software (Bio-rad).
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