DPCs (4000 cells/well) were seeded in a 96-well plate, cultured in medium for 24 h, and treated with vehicle (DMSO) as a control, 6.25, 12.5, 25, 50, and 100 μM of α-phellandrene dose-dependently, or 10 μM of forskolin. After 24, 48, or 72 h of treatment, the cells were incubated with the WST-1 solution (Sigma-Aldrich) diluted 1:10 in culture medium for 3 h. The absorbance was measured at 450 nm against a blank (without cells) using an M200 microplate reader (Tecan; Männedorf, Switzerland). To investigate the mechanism underlying the proliferative effect of α-phellandrene, adenylyl cyclase inhibitor SQ22,536 (50 μM, Sigma-Aldrich), PKA inhibitor H89 (10 µM, Sigma-Aldrich), or CREB inhibitor 666-15 (100 nM; Selleckchem; Houston, TX, USA) was added 1 h before 12.5 μM of α-phellandrene treatment. The proliferation percentage of cells was relatively determined by considering vehicle-only treated cells (control) as 100%.
Pka inhibitor h89
Manufactured by Merck Group
Sourced in United States
PKA inhibitor H89 is a laboratory reagent used for research purposes. It acts as a potent and selective inhibitor of protein kinase A (PKA). PKA is a serine/threonine protein kinase that plays a crucial role in various cellular signaling pathways. H89 can be utilized in experimental settings to study the effects of PKA inhibition on cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
P38 inhibitor sb203580, by Merck Group (2 mentions)
Restriction endonuclease, by New England Biolabs (2 mentions)
Anti pgc 1α, by Santa Cruz Biotechnology (2 mentions)
Anti tim23, by BD (2 mentions)
Pi3k inhibitor ly294002, by Merck Group (2 mentions)
Anti chchd4, by Proteintech (2 mentions)
Anti ha tag clone 3f10, by Roche (2 mentions)
Anti β actin and anti alpha tubulin, by Merck Group (2 mentions)
Anti hsp60, by Enzo Life Sciences (2 mentions)
Pkc inhibitor go6983, by Merck Group (2 mentions)
M200 microplate reader, by Tecan (1 mentions)
Wst 1 solution, by Merck Group (1 mentions)
Brain derived neurotrophic factor (bdnf), by Abcam (1 mentions)
Kt5823, by Cayman Chemical (1 mentions)
Bay 60 7550, by Merck Group (1 mentions)
Forskolin, by Merck Group (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions)
Atm inhibitor ku55933, by Merck Group (1 mentions)
Lantus solostar, by Sanofi (1 mentions)
11 protocols using pka inhibitor h89
1
Proliferative Effects of α-Phellandrene
2
Antibody Reagents for Western Blot Analysis
3
Pharmacological Modulation of Alzheimer's Pathology
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Aβ1–42 was obtained from rPeptide. Bay 60-7550 (CAS: 439083-90-6) was obtained from Sigma-Aldrich. The mice were treated with different doses of Bay 60-7550 (0.5, 1.0, 3.0 mg/kg/day, i.p.) or vehicle for 14 days after microinjection of Aβ1–42. PKG inhibitor KT5823 (Cayman Chemical, United States) and PKA inhibitor H89 (Sigma-Aldrich, United States) were microinjected bilaterally into the cerebroventricular, 30 min prior to treatment with Bay 60-7550. Antibodies against CRF, GR, p-CREB, CREB, and BDNF were obtained from Abcam (Cambridge, MA, United States). All secondary antibodies were obtained from Beyotime Biotechnology (Haimen, China).
4
Antibody Reagents for Western Blot Analysis
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Antibodies: anti-HA tag (clone 3F10) (Roche Applied Science), anti-β-actin and anti-alpha-tubulin (Sigma-Aldrich), anti-Smac (Upstate Cell Signaling Solutions, Lake Placid, NY), anti-Hsp60 (Enzo Life Sciences, Plymouth Meeting, PA), anti-VDAC1 (Calbiochem, Darmstadt, Germany), anti-GAPDH, anti-PDK4 and anti-Vinculin (Santa Cruz), anti-PGC-1α (a gift from Dr. Daniel P. Kelly, Sanford-Burnham Medical Research Institute, FL), anti-pAMPK, anti-pCREB and anti-CREB (Cell Signaling Technologies, Boston, MA), anti-CHCHD4 (Protein-Tech Group, IL), anti-Tim23 (BD Biosciences, San Diego, CA). The peroxidase-conjugated goat anti-rat, goat anti-rabbit, goat anti-mouse and horse anti-goat antibodies were from Vector Laboratories (Burlingame, CA). Rabbit polyclonal antibodies specific for human CHTM1 were generated in our laboratory through ProSci Inc. (Poway, CA) using full-length recombinant human CHTM1 protein purified from Escherichia coli. For cell transfections, Mirus (Madison, WI) and Lipofectamine 2000 (Invitrogen, Carlsbad, CA) were used. Restriction endonucleases were from New England BioLabs (Ipswich, MA). PKA inhibitor-H89, p38 inhibitor-SB203580, PI3K inhibitor-LY294002 and PKC inhibitor-GO6983 were from Sigma-Aldrich (St. Louis, MO). Other chemical reagents were from Thermo Fisher Scientific and Sigma-Aldrich.
5
Embryonic Fibroblast and Hepatocyte Isolation
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Mouse embryonic fibroblasts (MEFs) were isolated from 14.5-day CREB+/+ and CREBS111A embryos. Where indicated, primary MEFs were immortalized by transfection with a plasmid encoding with SV40 large T antigen (p129; a gift from Dr Janet Mertz). Primary hepatocytes were isolated from CREB+/+ and CREBS111A 12-weeks female mice using the collagenase perfusion method. Primary MEFs, immortalized MEFs, primary hepatocytes, HeLa and HEK 293T cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Forskolin (10 μM, Sigma) and IBMX (100 μM, Sigma) were treated for 90 min after serum starvation for overnight. PKA inhibitor H89 (10 μM, Sigma) and ATM inhibitor KU-55933 (10 μM, Sigma) were maintained in dimethyl sulfide (DMSO) and added to cells 30 min prior to stimulation. Calicheamicin A (CLM) was a kind gift of the Pfizer Compound Transfer Program and was maintained 4 μM stock solution in DMSO.
6
Regulation of Vascular Cell Dysfunction
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RGECs were cultured in 96‐well plates for 24 h, followed by a pre‐incubation with PKC‐β specific inhibitor LY‐333531 (10 nmol/L; Axon Ligands, Groningen, the Netherlands), PKC agonist phorbol‐myristate‐acetate (PMA; 10 μmol/L; Sigma), PKA inhibitor H‐89 (10 μmol/L; Sigma) or PKA agonist 8‐bromo‐adenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP; 100 μmol/L; Sigma) for 30 min. Then RGECs were treated with AGEs (200 μg/mL; Abcam), AGEs (200 μg/mL) + insulin (1 IU/mL, Lantus Solostar, Sanofi, Beijing, China) or AGEs (200 μg/mL) + rhGLP‐1 (1.0 mg/mL; Shanghai Benemae Pharmaceutical Corporation) for another 24 h according to a previously used method detect of reactive oxygen species (ROS) production16 .
RGECs were seeded in 24‐well plates and pre‐incubated with PKC inhibitor LY‐333531, PKC agonist PMA, PKA inhibitor H‐89 or PKA agonist 8‐Br‐cAMP for 30 min, and then treated with AGEs (200 μg/mL), AGEs (200 μg/mL) + insulin or AGEs (200 μg/mL) + rhGLP‐1 for 24 h. Detection of NO production followed a previous study16 .
RGECs were seeded in 24‐well plates and pre‐incubated with PKC inhibitor LY‐333531, PKC agonist PMA, PKA inhibitor H‐89 or PKA agonist 8‐Br‐cAMP for 30 min, and then treated with AGEs (200 μg/mL), AGEs (200 μg/mL) + insulin or AGEs (200 μg/mL) + rhGLP‐1 for 24 h. Detection of NO production followed a previous study
7
Phagocytic Function of Resident Peritoneal Macrophages
8
ABA and PKA Inhibitor Administration
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(±)-Cis,trans-ABA and PKA inhibitor (H.89) were purchased from Sigma-Aldrich (USA). ABA was dissolved in dimethyl sulfoxide (DMSO), then diluted with artificial cerebrospinal fluid (aCSF). The ratio of aCSF to DMSO was 2:1 (v/v). PKA inhibitor was dissolved in distilled water. These drugs were given in the volume of 10 µl (i.t.).
9
Cell signaling regulatory protocol
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LiCl, adenosine 5′-triphosphate (ATP) disodium salt hydrate, Akt inhibitor VIII, 8-bromoadenosine 3′,5′-cyclic monophosphate (8-br-cAMP), PKA inhibitor H89, and 6-bromo-indirubin-3′-oxime (BIO) were purchased from Sigma-Aldrich Korea. Tetramethyl-6-carboxyrhodamine (TAMRA)-labeled peptides (T-Pep, TAMRA-KEEPPSPPQSPR; T-Pep(p), TAMRA-KEEPPSPPQp-SPR) were synthesized by Peptron, Inc. ECL Prime Western Blotting Detection Reagent was obtained from Amersham Bioscience (RPN2232). Xpert protease and phosphatase inhibitor cocktail were purchased from GenDEPOT. Calyculin A was purchased from Abcam (ab141784).
10
Phagocytosis Assay of Macrophages
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