Endothelial cells were lysed in CETi lysis buffer (TransLab, Daejeon, Korea) on ice for 30 min and centrifuged at 13,000 rpm for 15 min. Supernatants were collected, and protein concentrations were determined at 595 nm using a protein assay kit (Pro-Measure, iNtRON Biotechnology, Seongnam, Gyeonggi, Korea). Equal amounts of total cellular proteins were boiled for 5 min and subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred onto nitrocellulose membranes and incubated with primary antibodies followed by incubation with anti-mouse or anti-rabbit secondary antibody as appropriate. Finally, the densities of protein bands were measured using an enhanced Hisol ECL Plus Detection Kit (BioFact, Daejeon, Korea). Antibodies against p-eNOS, p-CaMKII, p-AMPK, and p-CaMKKβ, as well as anti-mouse and anti-rabbit IgG antibodies, were purchased from Cell Signaling Technology (Beverly, MA, USA). An antibody against β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
P camkkβ
Manufactured by Cell Signaling Technology
Sourced in United States
P-CaMKKβ is a phospho-specific antibody that recognizes the autophosphorylated form of CaMKKβ (calcium/calmodulin-dependent protein kinase kinase beta). CaMKKβ is a serine/threonine protein kinase that plays a critical role in the calcium/calmodulin-dependent protein kinase (CaMK) signaling cascade.
Automatically generated - may contain errors
Lab products found in correlation
P ampk, by Cell Signaling Technology (4 mentions)
P camkii, by Cell Signaling Technology (2 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
P mtor, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
An antibody against β actin, by Santa Cruz Biotechnology (1 mentions)
Hisol ecl plus detection kit, by Biofact (1 mentions)
Antibodies against p enos, by Cell Signaling Technology (1 mentions)
Anti mouse and anti rabbit igg antibodies, by Cell Signaling Technology (1 mentions)
Pro measure, by iNtRON Biotechnology (1 mentions)
Grp78, by Cell Signaling Technology (1 mentions)
P ulk1, by Cell Signaling Technology (1 mentions)
β tublin, by Cell Signaling Technology (1 mentions)
Interleukin 6 (il 6), by Cell Signaling Technology (1 mentions)
Tnf α, by Cell Signaling Technology (1 mentions)
Il 1β, by Cell Signaling Technology (1 mentions)
Ire1α, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
5 protocols using p camkkβ
1
Endothelial Cell Protein Analysis
2
Regulation of INS-1 Cell Signaling
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INS-1 cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Foetal bovine serum (FBS; 16000-044) and RPMI-1640 culture medium were purchased from Hyclone (Logan, UT). Primary antibodies against PDX1, PERK, IRE1α, ATF6, GRP78, CaMKKβ, p-CaMKKβ, AMPK, p-AMPK, mTOR, p-mTOR, ULK1, p-ULK1, p62, Beclin 1, LC3, TNF-α, IL-1β, IL-6, Bax, Bcl-2, CASPASE3, GAPDH, and β-tublin were purchased from Cell Signalling Technology, Inc. (Danvers, MA, USA), as were secondary antibodies against rabbit immunoglobulin G (IgG) (STAR208P) and mouse IgG (STAR117P). Flou-4 AM, acridine orange (AO) dye, and ELISA kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). Total protein of treated cells was prepared using RIPA lysis buffer (Beijing Solarbio Science & Technology Co. Ltd., Beijing, China). Protein concentration was determined using a BCA protein quantitation kit (Thermo Co. Ltd., MA).
3
Western Blot Analysis of Signaling Pathways in RAW 264.7 Cells
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RAW 264.7 cells were lysed with RIPA reagents (Pierce, Rockford, IL, USA) containing cocktail protease and phosphatase inhibitors (Roche, Switzerland). Plasma proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). Blots were blocked with 5% BSA for 1 h and incubated with primary antibodies (1 : 1000 dilutions) for CaMKKβ, pCaMKKβ, AMPKα, pAMPKα, SIRT1, IκBα, IRF3, pIRF3, p38, p-p38, and tubulin (Cell Signaling, USA) at 4°C overnight. Then, the blots were further incubated with HRP-conjugated secondary IgG antibodies (1 : 2000 dilutions, Cell Signaling) at 37°C for 1 h. Chemiluminescence images were developed with SuperSignal Sensitivity Substrate Kit (Pierce) with a ChemiDoc XRS imaging system (Bio-Rad, USA).
4
Anti-inflammatory Signaling Pathways
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IPA (Figure 1 A) was provided by Dr. Young-Ho Kim (Chungnam National University, Daejeon, Korea). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin, and trypsin were purchased from Welgene (Gyeongsan, Korea). STO-609, zinc protoporphyrin IX (ZnPP), ML385, and LPS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Compound C were obtained from Tocris (Cookson, Bristol, UK), and EDTA was purchased from GenDEPOT (Barker, TX, USA). W7 was obtained from Calbiochem (La Jolla, CA, USA). Antibodies against p-NF-κB p65, p-IκB, IκB, p-AMPK, p-CaMKKβ, p-GSK3β(Ser9), and p-LKB1 were from Cell Signaling Technology (Beverly, MA, USA). Antibodies against NF-κB p65, HO-1, NOQ1, Nrf2, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Lamin B1 was purchased from Bioss Antibody, Inc. (Woburn, MA, USA). Tetrazole 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was acquired from USB Corporation (Cleveland, OH, USA). The cytotoxicity assay kit was obtained from Roche Applied Science (Indianapolis, IN, USA). All other chemicals were of the highest grade commercially available.
5
Calcium Signaling Pathways in Cellular Stress
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Ang II, Calhex 231 , R568, GdCl 3 , compound C, and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for calcium/calmodulin-dependent protein kinase II (CaMKII), p-CaMKII, p62, Beclin1, microtubule-associated protein light chain 3 (LC3), caspase-3, AMP-activated protein kinase (AMPK), p-AMPK, Ca 2+ /calmodulin-dependent-protein kinase-kinase-β (CaMKK β ), p-CaMKK β , mammalian target of rapamycin (mTOR), p-mTOR, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-CaSR was from Alpha Diagnostic International Inc. (San Antonio, TX, USA). The secondary antibody (alkaline phosphatase-conjugated anti-rabbit IgG) was from Promega Corporation (Madison, WI, USA). Polyvinylidene difluoride membranes were from Whatman (Buckinghamshire, UK).
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