Journal: The Journal of Neuroscience

Article Title: Activation of Presynaptic cAMP-Dependent Protein Kinase Is Required for Induction of Cerebellar Long-Term Potentiation

doi: 10.1523/jneurosci.19-23-10221.1999

Figure Lengend Snippet: Figure 2. PKA inhibitor drugs block cerebellar LTP induction in granule cell–Purkinje neuron pairs when applied in the bath but not in patch pipettes. A, PKA inhibitors KT5720 (10 mM) and Rp-8CPT-cAMP-S (100 mM) and the PKG inhibitor KT5823 (10 mM) were applied in the bath starting at t 5 217.5 min, as indicated by the thin horizontal bar. The control group in this graph is the same as the 10 mM BAPTA group from Figure 1 and is reproduced here for comparison. B, PKA inhibitors Rp-cAMP-S, Rp-8CPT-cAMP-S, and KT5720 (all 1 mM) were included in the patch pipette saline of either the granule cell or Purkinje neuron. The time of initiating whole-cell recording was at t 5 15 min (or slightly earlier), as indicated by the thin horizontal bar. C, Rp-cAMP-S (2 mM) was included in the granule cell patch pipette saline with an extended baseline

Article Snippet: KT5720 and KT5823 were purchased from Calbiochem (La Jolla, CA), Rp-8CPT-cAMP-S and Sp-8CPT-cAMP-S from Biolog (Hayward, CA), tetrodotoxin from Alexis Biochemicals (San Diego, CA), QX-314-Br from Alomone Labs (Jerusalem, Israel), Cs4-BAPTA from Molecular Probes (Eugene, OR), and all other compounds from Sigma (St. Louis, MO).

Techniques: Blocking Assay, Control, Comparison, Transferring, Saline

Journal: PLoS ONE

Article Title: The Effects of the Endocannabinoids Anandamide and 2-Arachidonoylglycerol on Human Osteoblast Proliferation and Differentiation

doi: 10.1371/journal.pone.0136546

Figure Lengend Snippet: (A) RT-PCR analysis of cannabinoid receptor mRNA expression in HOBs. Human astrocytes (HA) were used as a positive control for CB1R, CB2R and TRPV1 expression. Hypoxanthine-guanine PhosphoRibosylTransferase (HPRT) was used as the control house-keeping gene. (B) ALP production, normalised to DNA content, was measured in HOBs grown for 4 days and treated with 10μM anandamide (AEA) only, or 10μM anandamide and the following antagonists and inhibitors. Cells were treated with AM251 (100nM, CB 1 R antagonist), AM630 (100nM, CB 2 R antagonist) or Capsazepine (1μM, Cap, TRPV1 antagonist) for 30 minutes before the addition of vehicle or anandamide. (C) Cells were treated with KT5720 (500nM, PKA inhibitor), PD98059 (1μM, MEK inhibitor) or SP600125 (10μm, JNK inhibitor) for 30 minutes before the addition of vehicle or anandamide. Data given as mean ± S.E.M., n = 9, from 3 experiments. * P <0.05, ** P <0.01, compared to antagonist/inhibitor only; one-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: HOBs were treated with media supplemented with 100nM AM251 (CB 1 R antagonist), 100nM AM630 (CB 2 R antagonist), 1μM capsazepine (TRPV1 antagonist), 500nM KT5720 (PKA inhibitor, PKA is upstream of CREB), 1μM PD98059 (MEK inhibitor) or 10μm SP600125 (JNK inhibitor) for 30 minutes before the addition of vehicle (0.1% ethanol) or 10μM anandamide (all Tocris, UK).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control, Control

Journal: Nature Communications

Article Title: Cdk5 and GSK3β inhibit fast endophilin-mediated endocytosis

doi: 10.1038/s41467-021-22603-4

Figure Lengend Snippet: a Scoring criteria used in the kinase screen. Representative images of decreased, normal, and increased FEME in resting human RPE1 cells treated with 10 μM dobutamine, 10 μM DMSO, and 10 nM GDC-0941 (PI3Ki), respectively. Arrowheads point at FEME carriers. Decreased FEME was assigned for samples with >80% reduction in the number of cytoplasmic Endophilin-positive assemblies (EPAs), in at least 50% of the cells. Increased FEME was attributed to samples with >200% elevation in the number of EPAs, in at least 50% of the cells. The corresponding scoring marks were 0, 1, and 2, respectively. Scale bar, 5 μm. b Kinase screen using small compound inhibitors. RPE1 cells grown in complete medium were incubated for 10 min at 37 °C with the following inhibitors: DMSO, (vehicle); dobutamine, 10 μM (positive control); Dinaciclib (Cdk1/2/5/9i), 1 μM; CHIR-99041 (GSK3i1), 1 μM; BIO (GSK3i2), 1μM; Roscovitine (Cdk1/2/5i), 1 μM; PHA-793887 (Cdk2/5/7i), 100 nM; VX-745 (p38i), 10 μM; JNK-IN-8 (JNKi), 1 μM; staurosporine (broad kinases), 1 μM; GNE-7915 (LRRK2i), 1 μM; AZ191 (DYRK1Bi), 10 μM; GSK2334470 (PDKi), 10 μM; PF-4708671 (p70S6Ki), 10μM; AZ191 (DYRKi), 10 μM; AZD0530 (broad SRCi), 1 μM; TAK-632 (panRAFi), 10 μM; GW 5074 (CRAFi), 1 μM; PD0332991 (Cdk4/6i), 1 μM; MK2206 (AKTi), 1 μM; GDC-0879 (BRAFi), 1 μM; CX-4945 (CK2i), 1 μM; ZM 447439 (AurA/AurBi), 1 μM; RO-3306 (Cdk1i), 100 nM; BI 2536 (PLKi), 1 μM; PD0325901 (MEKi), 100 nM; Genistein (Y-kinases), 1 μM; Purvalanol A (Cdk1/2/4i), 100 nM; MLR 1023 (LYNi), 1 μM; P505-15 (SYKi), 100 nM; CDK1/2 inhibitor III (Cdk1/2i), 100 nM; KT 5720 (PKAi), 100 nM; BI-D1870 (p90RSKi), 100 nM; D4476 (CK1E), 1 μM; PF-4800567 (CK1Ei), 1 μM; SCH772984 (ERKi), 100 nM; STO609 (CaMKK1/2ii), 100 nM; P505-15 (SYKi), 1μM; PND-1186 (FAKi), 100 nM; Torin 1 (mTORC1/2i), 10 μM and GDC-0941 (PI3Ki), 100 nM (negative control). Histograms show the mean ± SEM from 12 well per condition, from three independent biological experiments. Statistical analysis was performed by one-way ANOVA. ns non significant; * P < 0.05, ** P < 0.01. c Number of FEME carriers (EPAs) upon titration of CHIR-99021, BIO, Roscovitine and Dinaciclib. Dobutamine and GDC-0941 were used as positive and negative controls, respectively. Plots show the mean ± SEM from three cells per condition and per timepoint, from three independent biological experiments. d β1-adrenergic receptor (β1AR) uptake into FEME carriers in RPE1 cells pre-treated with 5 μM CHIR-99021 (GSK3i) for 5 min, followed by 10 μM dobutamine for 4 min or not (resting). Scale bars, 5 μm. Histograms show the mean ± SEM of the number of FEME carriers (LHS: left hand side) and the number of FEME carriers positive for β1AR per 100 μm 2 (RHS: right hand side) ( n = 30 cells per condition, from biological triplicates). Arrowheads point at FEME carriers. Statistical analysis was performed by two-way ANOVA. ns non significant; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following small compound inhibitors (amongst the best-reported inhibitors for each kinase , ) were used: AZ191 (called DYRKi in this study Cayman 17693), AZD0530 aka Sacratinib (called SRCi in this study, Cayman 11497), BI-D1870 (called p90RSKi in this study, Cayman 15264), BIO-6-bromoindirubin-3′-oxime, aka BIO (called GSK3i2 in this study, (Sigma B1686), BI 2536 (called PLKi in this study, Selleckchem S1109), CDK1/2 inhibitor III (called Cdk1/2i in this study, Merck 217714), CHIR-99041 (called GSK3i1 in this study, Cayman 13122), Ciliobrevin D (called Ciliobrevin in this study, Calbiochem 250401), CX-4945 (called CK2i in this study, Cayman 16779), Dinaciclib (called Cdk1/2/5/9i in this study, MedChemExpress Hy-10492), Dobutamine (Sigma D0676), D4476 (called CK1i in this study, BioVision 1770), GDC-0879 (called BRAFi in this study, Tocris 4453), GDC-0941 (called PI3Ki in this study, Symansis SYG0941), Genistein (called Y-kinases in this study, Calbiochem 245834), GNE-7915 (called LRRK2i in this study, MedChemExpress Hy-10328), GSK2334470 (called PDKi in this study, Cayman 18095), GW 5074 (called CRAFi in this study, Santa Crux sc-200639), Harmine hydrochloride (called DYRKi in this study, Santa Crux sc2595136), JNK-IN-8 (called JNKi in this study MedChemExpress Hy-13319), KT 5720 (called PKAi in this study Cayman 10011011), MK2206 (called AKTi in this study, LKT Laboratories M4000), MLR 1023 (called LYNa in this study, Tocris 4582), PD0325901 (called MEKi in this study, Tocris 4192), PD0332991 aka Palbociclib (called Cdk4/6i in this study, Sigma PZ0199), PF-4708671 (called p70S6Ki in this study, MedChemExpress Hy-15773), PF-4800567 (called CK1Ei in this study, Cayman 19171), PHA-793887 (called Cdk2/5/7i in this study, ApexBio A5459), PND-1186 (called FAKi in this study, MedChemExpress Hy-13917), Purvalanol A (called Cdk1/2/4i in this study, Santa Cruz sc-224244), P505-15 (called SYKi in this study, Adooq Bioscence A11952), Roscovitine (called Cdk1/2/5i in this study, Santa Cruz sc-24002), RO-3306 (called Cdk1i in this study, Cayman 15149), SCH772984 (called ERKi in this study, Sellekchem S7101), Staurosporine (called broad kinases in this study, Alomone Labs AM-2282), STO609 (called CaMKK1/2i in this study, Cayman 15325), TAK-632 (called panRAFi in this study, Selleckchem S7291), Torin 1 (called mTORC1i in this study, Tocris 4247), VX-745 (called p38i in this study, MedChemExpress Hy-10328) and ZM 447439 (called AurA/AurBi in this study, Cayman 13601).

Techniques: Incubation, Positive Control, Negative Control, Titration

Journal: The American Journal of Pathology

Article Title: A Role for cAMP and Protein Kinase A in Experimental Necrotizing Enterocolitis

doi: 10.1016/j.ajpath.2016.10.014

Figure Lengend Snippet: A: PKA inhibitor (KT5720) inhibits caspase-3 activation and PKA activation in vitro. After 4 to 6 hours of infection both caspase-3 activation and pPKA were significantly increased. IEC-6 cells were treated with 1 μmol/L of KT5720 before exposure to CS demonstrated significantly less caspase-3 and PKA activation after 6 hours. B: CS-induced apoptosis is decreased by pharmacologic PKA inhibition. Immunofluorescence of DNA fragmentation and apoptosis were significantly decreased in the presence of CS even after 6 hours of infection when cells were pretreated with PKA inhibitors (KT5720, Rp-8-br-cAMPS, PKI) compared with those cells that received CS alone. The greatest decrease occurred in those cells treated with KT5720 compared with other inhibitors. IEC-6 cells were grown on chamber slides, and after various doses and exposure times they were fixed and stained with ApoTag for apoptosis and DAPI (blue). Apoptotic cells were counted per 1000 cells. C: Knockdown of PKA showed a significant reduction in caspase-3 cleavage. IEC-6 cells were transfected with lipofectamine 2000 and siRNA for PKA. Cells were then treated for 6 hours with CS. Western blot analysis of cleaved caspase-3, PKA, and β-actin were performed. PKA knockdown was confirmed with a significant reduction in PKA expression. A significant reduction in cleaved caspase 3 was also seen at the 6-hour time point. D: Knockdown of PKA protects IEC-6 cells from CS-induced apoptosis. IEC-6 cells were transfected with lipofectamine 2000 and siRNA for PKA. Cells were then treated over a time course with CS. There was a significant increase in apoptosis in the presence of CS after 6 hours. In addition, ApoTag staining revealed fewer apoptotic cells when PKA was knocked down (+siRNA) before infection with CS for 6 hours than controls (−siRNA) (P = 0.0014). ∗P < 0.05, ∗∗∗P < 0.001, and ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. Original magnification, ×10 (B and D). CS, Cronobacter sakazakii; IEC-6, rat intestinal epithelial cell line; KD, knockdown; PKA, protein kinase A; PKI, protein kinase inhibitor; pPKA, PKA phosphorylation: WT, wild-type.

Article Snippet: Cells were pretreated with PKA inhibitors (0.1–20 μmol/L) KT5720 (Cayman Chemical, Ann Arbor, MI), cAMP Dependent Protein Kinase Inhibitor (Sigma-Aldrich, St. Louis, MO), Rp-8-cAMPS (Santa Cruz Biotechnology, Dallas, TX) for 30 minutes then subjected to co-culture with C. sakazakii at various concentrations over a time course.

Techniques: Activation Assay, In Vitro, Infection, Inhibition, Immunofluorescence, Staining, Knockdown, Transfection, Western Blot, Expressing, Phospho-proteomics

Journal: PLoS Computational Biology

Article Title: Temporal Sensitivity of Protein Kinase A Activation in Late-Phase Long Term Potentiation

doi: 10.1371/journal.pcbi.1000691

Figure Lengend Snippet: (A) LTP induced using a 80 sec inter-train interval (n = 7) is blocked by KT5720 ( p <0.05), which demonstrates PKA dependence. (B) In contrast, when using a 40 sec inter-train interval (bottom; n = 7), LTP in KT5720 slices is not different from vehicle control ( p >0.05), demonstrating PKA independence. In both panels, fEPSPs for KT5720 treated slices are shown with red circles, and vehicle controls with blue circles. (C) Summary of temporal sensitivity of PKA dependence of L-LTP. Data for 3 sec, 20 sec and 300 sec are from .

Article Snippet: KT5720 (Biomol), an inhibitor of catalytic subunits of PKA , was dissolved in DMSO and diluted in ACSF to a final perfusate concentration of 1 μM (0.1% DMSO), the control slices were pretreated with 0.1% DMSO vehicle, which had no effect on basal fEPSPs.

Techniques: Control

Journal: The Journal of Neuroscience

Article Title: Isoform-Specific Regulation of the Na + /Ca 2+ Exchanger in Rat Astrocytes and Neurons by PKA

doi: 10.1523/JNEUROSCI.18-13-04833.1998

Figure Lengend Snippet: PKA activation increases Ca2+uptake in oocytes expressing the neuron NCX1 isoform but not an astrocyte isoform. A,45Ca2+ uptake was measured inXenopus oocytes injected with cRNA of neuronal isoform AD. Oocytes were exposed to 90 mm NaCl or 90 mmKCl (0 Na); 5 mm NiCl2 was used to block the exchanger. Ca2+ uptake in 0 Na+solution was significantly higher than that in 90 mmNa+ solution (p < 0.001) and was blocked by Ni2+. After PKA activation (described in Materials and Methods), Ca2+ uptake was significantly higher than that in 0 Na+ only (p < 0.001), and this upregulation was blocked by KT5720 (p < 0.001).B, 45Ca2+ uptake was measured in Xenopus oocytes injected with cRNA of astrocyte isoform BD. Ca2+ uptake in 0 Na+ was significantly higher than that in 90 mm Na+ (p < 0.001) and was blocked by Ni2+. But in contrast, activation by PKA did not increase the Ca2+ uptake in 0 Na+. Values are mean ± SEM (n = 10–25 oocytes). An A exon-containing isoform (AD) found in neurons is upregulated after activation of the PKA pathway, whereas isoform BD, found in astrocytes, is not.

Article Snippet: For PKA inhibition, the oocytes were preincubated in 90 m m Na + solution containing 1 μ m KT5720 (LC Laboratories) ( Kamei et al., 1992 ) for 2 hr before the oocytes were switched to PKA-activating cocktail.

Techniques: Activation Assay, Expressing, Injection, Blocking Assay