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  • 93
    Millipore h89
    Effects of PKA inhibition on the osthole-induced suppression of p38 phosphorylation in LPS-stimulated cells. Raw264.7 cells were incubated with 50 μmol/L osthole for 24 h. Cells were then treated with 10 μmol/L <t>H89</t> or 1 μmol/L KT5720 for 2 h before exposure to 1 μg/mL LPS. Western blot analysis was performed 30 min after stimulation with LPS.
    H89, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 2144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore h 89 dihydrochloride hydrate
    Effect of protein kinase A <t>inhibitor—H-89</t> (10 −5 M) on basal and ACTH (10 −7 M) stimulated Giot1 gene expression in primary culture of rat adrenocortical cells. ( A ) The expression of Giot1 was determined by qPCR method. Cells were collected for RNA isolation 2 and 24 h following administration of the tested compounds. Bars represent means ± SEM ( n = 5); ( B ) effect of H-89 (10 −5 M) on basal and ACTH (10 −7 M) stimulated corticosterone output in primary cultures of rat adrenocortical cells. Cell culture media were collected at 2 and 24 h after administration of the tested compounds. Corticosterone concentration was determined by ELISA. Data are presented as means ± SEM ( n = 6). Statistical analysis of the data was performed by using one-way analysis of variance (ANOVA) followed by Tukey’s HSD test. Groups sharing the same letter are not significantly different according to Tukey’s HSD test.
    H 89 Dihydrochloride Hydrate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    h89  (Tocris)
    94
    Tocris h89
    Deletion of PfPDEβ bypasses the need for PKG activity upstream of MyoA S 19 phosphorylation in mature schizonts and results in premature proteolytic shedding of AMA1. (A) PKG-independent phosphorylation of MyoA in PfPDEβ-null parasites. Western blot of PKG inhibitor–treated (+C2) and E64-treated PfPDEβ ΔcatHA schizont proteins from DMSO- and RAP-treated PfPDEβ ΔcatHA cultures, probed with a phospho-S 19 MyoA antibody. The blot was reprobed with a polyclonal anti-MyoA antibody to determine the relative amounts of MyoA present in each sample. The lower panel shows total protein levels in the gel prior to blotting. The schematic shows the effect of various compounds on egress: Compound 2 and BAPTA-AM block egress upstream of PVM rupture, BIPPO induces egress, and E64 prevents erythrocyte plasma membrane rupture. (B) The <t>PKA</t> inhibitor <t>H89</t> inhibits MyoA S 19 phosphorylation. Western blot analysis of E64-arrested wild-type schizonts (mock-treated PfPDEβ ΔcatHA ) treated with increasing concentrations of the PKA inhibitor H89. A sample was taken before inhibitor addition (start) and a Compound 2–blocked sample served as negative control. Blots were probed with antibodies against phospho-S 19 MyoA, total MyoA, and GAPDH as a loading control. (C) The PDE inhibitor BIPPO induces MyoA phosphorylation. Western blot analysis of Compound 2–arrested mock- or RAP-treated PfPDEβ ΔcatHA schizonts, following incubation with increasing concentrations of the PDE inhibitor BIPPO. A sample was taken before inhibitor addition to show the absence of MyoA S 19 phosphorylation (start). Blots were probed with the same antibodies as in (B). (D) BAPTA-AM inhibits MyoA phosphorylation. Effects of BAPTA-AM on MyoA S 19 phosphorylation in Compound 2–arrested (top panel) or E64-arrested (bottom panel) PfPDEβ ΔcatHA schizonts following mock or RAP treatment. Western blots were additionally probed with the same antibodies as in B and C. (E) Dual-stained IFAs showing the cellular distribution of AMA1 in RAP- and mock-treated (DMSO) PfPDEβ ΔcatHA schizonts. Schizonts were co-stained with an AMA1 antibody (red) and a HA antibody (green) to detect the presence or absence of PfPDEβ. DAPI (blue) was used to visualise nuclear material. Scale bar, 5 μm. The bar chart (right) shows quantitation of proportions of schizonts displaying apical or peripheral AMA1 staining in E64-arrested PfPDEβ ΔcatHA schizonts. Data presented are from a representative experiment in which > 50 schizonts per condition were counted by two researchers. Error bars, 1 SD. (F) Western blot analysis of culture supernatants from RAP- and mock-treated PfPDEβ ΔcatHA purified schizonts sampled over 90 minutes of culture. The blot was probed with antibodies specific for the micronemal proteins AMA1 and EBA175, as well as SERA5, to measure schizont rupture. Positions of molecular weight markers are indicated (left). The blot is representative of at least three independent experiments. (G) Quantitation of AMA1 shedding by IFA in merozoites released from RAP- and mock-treated PfPDEβ ΔcatHA schizonts. Purified merozoites were stained with an antibody against the AMA1 ectodomain. Thumbnails show examples for translocated (surface), shed (−), and micronemal (apical) AMA1 in green. Nuclei were stained with DAPI (blue). The bar charts show the mean proportions of merozoites exhibiting surface (left) and apical (right) AMA1 staining for each condition. More than 100 merozoites were counted by two researchers. Error bars, 1 SD. *, significant by unpaired t test ( p -value = 0.005); n.s., not significant ( p -value = 0.306). AMA1, apical membrane antigen-1; BAPTA-AM, 1,2-bis( o -aminophenoxy)ethane- N , N , N′ , N′ -tetraacetic acid-acetoxymethyl ester; BIPPO, 5-Benzyl-3-isopropyl-1 H -pyrazolo[4,3- d ]pyrimidin-7(6 H )-one; EBA175, erythrocyte-binding antigen 175; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HA, haemagglutinin; IFA, immunofluorescence assay; MyoA, myosin A; n.s., not significant; PDE, phosphodiesterase; PfPDEβ, Plasmodium falciparum phosphodiesterase β; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PVM, parasitophorous vacuole membrane; RAP, rapamycin; SERA5, serine repeat antigen 5.
    H89, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 227 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cayman Chemical h89
    YAP stabilizes phosphorylated CREB upon RA treatment. ( A ) Endogenous CREB was immunoprecipitated from WT and YAP KO cells after treatment with MG132 to block degradation and probed for ubiquitination (Ub). ( B ) Western blotting of CREB in cells treated with RA and <t>H89.</t> ( C ) Western blotting of p‐CREB and CREB after transiently expressed YAP in knockout cells. ( D ) Quantification of the percentage of neurite‐bearing cells and average neurite length in KO cells after transfected with YAP or empty vector. Data show the Mean ± S.E.M. values of three replicates (* P
    H89, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    LC Laboratories h 89
    Blockade of Protein Kinase A impairs T3's effect on β-catenin, Cyclin-D1 and hepatocyte proliferation in mice A. A representative western blot using pooled samples from three wells per condition (left) shows increased levels of pSer675-β-catenin and pSer133-CREB in primary mouse hepatocytes after 30 minutes of T3 treatment. Inclusion of PKA inhibitor <t>H89</t> (1 μM) in the media 30 minutes prior to the addition of T3 (100 nM) showed a notable decrease in pSer675-β-catenin and pSer133-CREB levels. Densitometry on the representative WB (right) shows an increase in pSer675-β-catenin and pSer133-CREB after T3 treatment, which was blocked by H89 treatment. (I.O.D. – integrated optical density). B. A representative western blot shows a decrease in the hepatic levels of pSer675-β-catenin and Cyclin-D1 when H89 was injected twice IP in 3-day T3 fed mice as compared to 3 day T3 only group. Gapdh verifies equal loading. Each lane represents an individual sample. C. A representative micrograph (200×) illustrates a decrease in the number of Cyclin-D1-positive hepatocytes when H89 was injected twice to the 3-day T3-fed mice as compared to T3 only group. Three or more mice per group were used for this study. D. Quantification of the Cyclin-D1-positive hepatocytes shows a significant decrease in positive cells in H89+T3 as compared to T3 only group (*p
    H 89, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 91/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH h89
    Proliferative focus morphology. Sertoli cells were treated for 24 h with retinol in the presence or absence of the antioxidant Trolox (100 μmol/L), the p38 inhibitor SB203580 (10 μmol/L), the JNK inhibitor SP600125 (10 μmol/L), the Akt inhibitor LY294002 (10 μmol/L), the ERK inhibitor U0126 (10 μmol/L) the pan-PKC inhibitor GÖ6983 (10 μmol/L) and the PKA inhibitor <t>H89</t> (1 μmol/L); medium was then replaced in all groups for 10% FBS-supplemented 199 medium without any other compound for 14 d, with exchange for fresh medium every 3 d. At the end of the 14 d period, morphology was examined in a phase-contrast microscope (representative micrographs at ×40 are depicted) and proliferative foci were counted in each well (see Figure 5A for scores).
    H89, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 108 article reviews
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    95
    Selleck Chemicals h 89 2hcl
    Proliferative focus morphology. Sertoli cells were treated for 24 h with retinol in the presence or absence of the antioxidant Trolox (100 μmol/L), the p38 inhibitor SB203580 (10 μmol/L), the JNK inhibitor SP600125 (10 μmol/L), the Akt inhibitor LY294002 (10 μmol/L), the ERK inhibitor U0126 (10 μmol/L) the pan-PKC inhibitor GÖ6983 (10 μmol/L) and the PKA inhibitor <t>H89</t> (1 μmol/L); medium was then replaced in all groups for 10% FBS-supplemented 199 medium without any other compound for 14 d, with exchange for fresh medium every 3 d. At the end of the 14 d period, morphology was examined in a phase-contrast microscope (representative micrographs at ×40 are depicted) and proliferative foci were counted in each well (see Figure 5A for scores).
    H 89 2hcl, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 95/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Enzo Biochem h89
    Phosphorylation at S675 in β-catenin E654 by protein kinase A (PKA). (A–C) Immunoblotting showing total β-catenin (βcat) and phosphoS675 β -catenin (pS675) expression levels in total lysates of wild-type (YY), β-catenin Y654/E654 (YE) and β-catenin E654/E654 (EE) mouse embryonic fibroblasts (MEF) (A), embryos (B) and intestinal tumours from Apc and Apc;YE mice (C). Tubulin (TUB) expression levels were used as a loading control. The relative expression levels were determined in three individual experiments and averaged. Numbers between brackets refer to the number of independent cell lines used for each genotype. (D, E) MEF were treated with <t>H89</t> (D) or forskolin (FSK) (E). Effects on PKA activity were evaluated by determining the expression levels of phosphoCREB (pCREB, upper band; lower band is phosphoATF-1). Following treatment, phosphoS675 β -catenin expression levels were determined and quantified. DMSO, dimethyl sulphoxide.
    H89, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 91/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Beyotime h89
    The traces of the 60 s spatial probe trials, the frequency of swimming across the platform site, and the swimming speed of the five groups. Cerebral hypoperfusion effected the spatial probe trial as demonstrated by the decreased frequency of swimming across the platform during the 60 s interval. In the EA group, the frequency increased (a); after ICV injection of <t>H89,</t> the increased frequency decreased (b), while there were no significant differences in the swimming speed either among the control, model, and EA groups or between the EA + NS and EA + H89 groups ((c) and (d)). The data are expressed as the mean ± SEM. ANOVA statistical analyses were performed to compare the means among the control, model, and EA groups; ※ P
    H89, supplied by Beyotime, used in various techniques. Bioz Stars score: 93/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Seikagaku h89
    FCS and FCCS analyses in live cells expressing DsRed-AQP2 and GFP-actin. (A and D) The crosshair indicates the measurement position: A, subapical region; D, apical membrane. Images for GFP-actin (green) and DsRed-AQP2 (red) were overlayed. In all images, the orthogonal z -stack profiles along the indicated lines in the x or y dimension of the central xy image are shown in the rectangular windows at the bottom (xz) and to the right (yz) of the central image. Bars, 5 μm. (B and E) FCCS analysis in the subapical region (B) and the apical membrane (E). Autocorrelation curves at the positions indicated in the images above are shown. Blue lines correspond to GFP-actin signal, red lines correspond to DsRed-AQP2 signal, and black lines are cross-correlation curves. The dashed lines are measured curves and the solid lines are their fitted curves. Insets show the fluorescence intensities of DsRed-AQP2 and GFP-actin in the two respective channels during an FCCS measurement. CR, count rates. (C and F) Cross-correlation curves before (Before FK) and after (After FK) forskolin stimulation. (G) Summary of relative cross-correlation amplitude ([ Gc (0)-1]/[ Gr (0)-1]). C, control; FK, forskolin treatment; <t>H89,</t> H89-pretreated cells expressing DsRed-AQP2 and GFP-actin; S256A, cells expressing DsRed-S256A-AQP2 and GFP-actin; subapical, measurements in the subapical region; apical, measurements on the apical membrane. Data represent the mean and SE from at least five independent experiments. *, P
    H89, supplied by Seikagaku, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cayman Chemical pka inhibitor h89
    S. aureus lipoproteins and host TLR2, TLR4, and NLRP3 are involved in secretion of PGE 2 and proinflammatory cytokines and chemokines by macrophages after S. aureus infection. WT and corresponding gene-deficient macrophages were infected with multiple S. aureus strains (SA113, Δ lgt , and + pRB) at MOI 10:1 or not infected. PGE 2 , TNF-α, IL-1β, and RANTES release into the supernatant of macrophage cultures was analyzed by ELISA 9 h after infection ( a–h ). Activation of the <t>cAMP-PKA</t> (P-PKA) and MAPK (P-ERK and P-p38) pathways was evaluated by western blotting. GAPDH served as a loading control ( i , j ). WT macrophages (untreated or pre-treated with 10 µM <t>H89</t> for 2 h) were infected with WT S. aureus strains at MOI 10:1 or not infected. TNF-α, IL-1β, and RANTES production in the supernatants of macrophages was analyzed by ELISA 9 h after infection ( k ). Results are expressed as the mean ± SD of 3 independent experiments and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test or 2-way ANOVA with Bonferroni's post-test. * p
    Pka Inhibitor H89, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzo Biochem pka inhibitor h89
    YC-1 activates cyclic nucleotide-dependent protein kinases in A549 cells. ( A ) A549 cells were pretreated with YC-1 for 1 h and incubated under normoxia or hypoxia for another 1 h. Cell lysates were subjected to Western blotting for phospho-VASP. ( B , C ) A549 cells were pretreated with the PDE inhibitor IBMX (100 μM), the sGC inhibitor ODQ (10 μM), or the <t>PKA</t> inhibitor <t>H89</t> (5 μM) for 30 min, then treated with YC-1 or BAY 41-2272 (BAY) for 1 h and incubated under hypoxia for another 24 h. TF expression was determined by Western blotting.
    Pka Inhibitor H89, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    h89  (Abcam)
    93
    Abcam h89
    The role of PKA and CK2 in regulating SK2 channels in intrinsic plasticity. A , Example traces for cells treated with <t>H89,</t> a PKA inhibitor that was present in the bath for the duration of the experiment. B , Time graph for changes in spiking relative to baseline. Either somatic depolarization or oxo-m was applied from minute 5 to 10. C , Example traces for cells treated with TBB, a CK2 inhibitor that was present in the bath for the duration of the experiment. D , Time graph for changes in spiking relative to baseline. Either somatic depolarization or oxo-m was applied from minute 5 to 10. E , Bar graph for depolarization-induced changes in spiking relative to baseline. Both H89 and TBB bath application prevented intrinsic plasticity. F , Bar graph for oxo-m-induced changes in spiking relative to baseline. H89, but not TBB, prevented intrinsic plasticity; * p
    H89, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Tocris pka inhibitor h 89
    Effect of the <t>PKA</t> inhibitor H-89 on agonist-induced ERK1/2 phosphorylation in D1R and D1R-D3R cells. ERK1/2 phosphorylation in CHO cells transfected with D1R-Rluc cDNA (1 μg cDNA) alone (A) or D1R-Rluc (1 μg cDNA) and D3R-YFP (1.3 μg cDNA) (B-D). 48 h post-transfection, D1R or D1R-D3R cells were pre-treated or not with the PKA inhibitor H-89 (10 μM for 30 min) and were treated for 7 min with the D1R agonist SKF 38393 (1 μM) (A-D), the D3R agonist PD 128907 (1 μM) (C-D) or both (C-D). Quantification of phosphorylated ERK1/2 was determined by Western blot. Representative blots are shown. Values are mean ± S.E.M. (n = 4 with duplicates) and are expressed as percentage of basal or non-treated cells; * , ** and ***: p
    Pka Inhibitor H 89, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    Standard format Plasmid sent in bacteria as agar stab
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    N/A
    Anti Langerin RABBIT Antibody 200 401 H89
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    Image Search Results


    Effects of PKA inhibition on the osthole-induced suppression of p38 phosphorylation in LPS-stimulated cells. Raw264.7 cells were incubated with 50 μmol/L osthole for 24 h. Cells were then treated with 10 μmol/L H89 or 1 μmol/L KT5720 for 2 h before exposure to 1 μg/mL LPS. Western blot analysis was performed 30 min after stimulation with LPS.

    Journal: Acta Pharmacologica Sinica

    Article Title: Osthole pretreatment alleviates TNBS-induced colitis in mice via both cAMP/PKA-dependent and independent pathways

    doi: 10.1038/aps.2017.71

    Figure Lengend Snippet: Effects of PKA inhibition on the osthole-induced suppression of p38 phosphorylation in LPS-stimulated cells. Raw264.7 cells were incubated with 50 μmol/L osthole for 24 h. Cells were then treated with 10 μmol/L H89 or 1 μmol/L KT5720 for 2 h before exposure to 1 μg/mL LPS. Western blot analysis was performed 30 min after stimulation with LPS.

    Article Snippet: In some experiments, H89 (10 mg/kg, Sigma, USA), a specific protein kinase A (PKA) inhibitor, was intraperitoneally administered 1 h prior to the TNBS treatment as indicated.

    Techniques: Inhibition, Incubation, Western Blot

    Effect of protein kinase A inhibitor—H-89 (10 −5 M) on basal and ACTH (10 −7 M) stimulated Giot1 gene expression in primary culture of rat adrenocortical cells. ( A ) The expression of Giot1 was determined by qPCR method. Cells were collected for RNA isolation 2 and 24 h following administration of the tested compounds. Bars represent means ± SEM ( n = 5); ( B ) effect of H-89 (10 −5 M) on basal and ACTH (10 −7 M) stimulated corticosterone output in primary cultures of rat adrenocortical cells. Cell culture media were collected at 2 and 24 h after administration of the tested compounds. Corticosterone concentration was determined by ELISA. Data are presented as means ± SEM ( n = 6). Statistical analysis of the data was performed by using one-way analysis of variance (ANOVA) followed by Tukey’s HSD test. Groups sharing the same letter are not significantly different according to Tukey’s HSD test.

    Journal: International Journal of Molecular Sciences

    Article Title: Effect of ACTH and hCG on the Expression of Gonadotropin-Inducible Ovarian Transcription Factor 1 (Giot1) Gene in the Rat Adrenal Gland

    doi: 10.3390/ijms19082285

    Figure Lengend Snippet: Effect of protein kinase A inhibitor—H-89 (10 −5 M) on basal and ACTH (10 −7 M) stimulated Giot1 gene expression in primary culture of rat adrenocortical cells. ( A ) The expression of Giot1 was determined by qPCR method. Cells were collected for RNA isolation 2 and 24 h following administration of the tested compounds. Bars represent means ± SEM ( n = 5); ( B ) effect of H-89 (10 −5 M) on basal and ACTH (10 −7 M) stimulated corticosterone output in primary cultures of rat adrenocortical cells. Cell culture media were collected at 2 and 24 h after administration of the tested compounds. Corticosterone concentration was determined by ELISA. Data are presented as means ± SEM ( n = 6). Statistical analysis of the data was performed by using one-way analysis of variance (ANOVA) followed by Tukey’s HSD test. Groups sharing the same letter are not significantly different according to Tukey’s HSD test.

    Article Snippet: ACTH (Synacthen) was purchased from Novartis (Basel, Switzerland), testosterone (Testoviron-Depot) from Schering AG (Berlin, Germany), estradiol (Estradiol-Depot) from Jenapharm (Jena, Germany) and H-89 dihydrochloride hydrate (Catalog Number B1427) and hCG (Chorionic gonadotropin human Catalog Number CG10, lyophilized powder) from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Protein kinase A (PKA)-dependent regulation of endogenous Kv7.5 currents in cultured HASMCs. ( A ) Representative current traces recorded in a single HASMC (Capacitance = 281 pF) before (i. control) and 5 min after addition of 10 µM retigabine (ii). ( B ) Mean fractional conductance plot calculated from steady-state endogenous Kv7 currents fitted to a Boltzmann distribution (V 0.5 = −40.8 mV, n = 10). ( C ) I–V relationships of Kv7 currents recorded in HASMCs before (control, filled circles, n = 5), after 5 min treatment with 1 µM forskolin (open circles, n = 4), and after 5 min treatment with diclofenac (100 µM, open triangles, n = 4). ( D ) I–V relationships of Kv7 currents recorded in HASMCs before (control, filled circles, n = 7), after 5 min treatment with 1 nM formoterol (open circles, n = 7), and after 5 min treatment with Kv7 channel blocker XE991 (1 µM) in the presence of 1 nM formoterol (closed triangles, n = 3). ( E ) Representative time-courses of 1 nM formoterol application recorded at −20 mV in a single untreated HASMC (black, Capacitance = 39 pF) and a HASMC pretreated with 10 µM H-89 (red, C = 127 pF). ( F ) Relative formoterol-induced enhancement of the current recorded at −20 mV in untreated HASMCs (black bars, n = 7) and in HASMCs pretreated with H-89 (10 µM for 20 min, red bar, n = 4). * Significant difference from control ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Mechanisms of PKA-Dependent Potentiation of Kv7.5 Channel Activity in Human Airway Smooth Muscle Cells

    doi: 10.3390/ijms19082223

    Figure Lengend Snippet: Protein kinase A (PKA)-dependent regulation of endogenous Kv7.5 currents in cultured HASMCs. ( A ) Representative current traces recorded in a single HASMC (Capacitance = 281 pF) before (i. control) and 5 min after addition of 10 µM retigabine (ii). ( B ) Mean fractional conductance plot calculated from steady-state endogenous Kv7 currents fitted to a Boltzmann distribution (V 0.5 = −40.8 mV, n = 10). ( C ) I–V relationships of Kv7 currents recorded in HASMCs before (control, filled circles, n = 5), after 5 min treatment with 1 µM forskolin (open circles, n = 4), and after 5 min treatment with diclofenac (100 µM, open triangles, n = 4). ( D ) I–V relationships of Kv7 currents recorded in HASMCs before (control, filled circles, n = 7), after 5 min treatment with 1 nM formoterol (open circles, n = 7), and after 5 min treatment with Kv7 channel blocker XE991 (1 µM) in the presence of 1 nM formoterol (closed triangles, n = 3). ( E ) Representative time-courses of 1 nM formoterol application recorded at −20 mV in a single untreated HASMC (black, Capacitance = 39 pF) and a HASMC pretreated with 10 µM H-89 (red, C = 127 pF). ( F ) Relative formoterol-induced enhancement of the current recorded at −20 mV in untreated HASMCs (black bars, n = 7) and in HASMCs pretreated with H-89 (10 µM for 20 min, red bar, n = 4). * Significant difference from control ( p

    Article Snippet: Amphotericin B, H-89 dihydrochloride were from Calbiochem.

    Techniques: Cell Culture

    Misoprostol opposes hypoxia-induced Bnip3-FL expression . a Immunoblot for Bnip3-FL in protein extracts from the hippocampus, large intestine, and heart of PND10 rat pups exposed to hypoxia (10% O 2 ) ± misoprostol for 7 days. b HCT-116 cells were transfected with PKA biosensor (pPHT-PKA), and treated with 10 μM misoprostol or vehicle for 2 h. Cells were imaged by standard fluorescence microscopy. c Quantification of fluorescent images in b by measuring the ratio of green (active) to red (inactive) fluorescent signal, normalized to cell area, quantified in 10 random fields. d HCT-116 cells were transfected with GFP-P65 (green) and were treated with 10 μM misoprostol ± 10 μM H89 for 20 h. Cells were stained with Hoechst (blue) and imaged by standard fluorescence microscopy. e Quantification of fluorescent images in d by calculating the percentage of cells with nuclear P65 signal over 10 random fields. f HCT-116 cells were transfected with IkBa(SS32,36AA) [indicated as IkB (S, A)] or an empty vector control. GFP-P65 (green) was used to indicate subcellular P65 localization in all conditions. Cells were treated with 10 μM misoprostol or vehicle control for 20 h. Cells were stained with Hoechst (blue) and imaged by standard fluorescence microscopy. g Quantification of fluorescent images in d by calculating the percentage of cells with nuclear P65 signal over 10 random fields. h HCT-116 cells were treated with 10 μM misoprostol or vehicle for 20 h, during extraction proteins were fractioned according to their sub-cellular compartment. Protein extracts were immunoblotted, as indicated. i 3T3 cells were treated with 10 μM misoprostol or vehicle control for 4 h. Protein extracts were immunoblotted as indicated. j HCT-116 cells were transfected with wild-type P65 or empty vector control. Extracts were immunoblotted, as indicated. k HCT-116 cells were transfected with P65 constructs for 20 h. Extracts were immunoblotted, as indicated. Data are represented as mean ± S.E.M. * P

    Journal: Cell Death Discovery

    Article Title: Misoprostol regulates Bnip3 repression and alternative splicing to control cellular calcium homeostasis during hypoxic stress

    doi: 10.1038/s41420-018-0104-z

    Figure Lengend Snippet: Misoprostol opposes hypoxia-induced Bnip3-FL expression . a Immunoblot for Bnip3-FL in protein extracts from the hippocampus, large intestine, and heart of PND10 rat pups exposed to hypoxia (10% O 2 ) ± misoprostol for 7 days. b HCT-116 cells were transfected with PKA biosensor (pPHT-PKA), and treated with 10 μM misoprostol or vehicle for 2 h. Cells were imaged by standard fluorescence microscopy. c Quantification of fluorescent images in b by measuring the ratio of green (active) to red (inactive) fluorescent signal, normalized to cell area, quantified in 10 random fields. d HCT-116 cells were transfected with GFP-P65 (green) and were treated with 10 μM misoprostol ± 10 μM H89 for 20 h. Cells were stained with Hoechst (blue) and imaged by standard fluorescence microscopy. e Quantification of fluorescent images in d by calculating the percentage of cells with nuclear P65 signal over 10 random fields. f HCT-116 cells were transfected with IkBa(SS32,36AA) [indicated as IkB (S, A)] or an empty vector control. GFP-P65 (green) was used to indicate subcellular P65 localization in all conditions. Cells were treated with 10 μM misoprostol or vehicle control for 20 h. Cells were stained with Hoechst (blue) and imaged by standard fluorescence microscopy. g Quantification of fluorescent images in d by calculating the percentage of cells with nuclear P65 signal over 10 random fields. h HCT-116 cells were treated with 10 μM misoprostol or vehicle for 20 h, during extraction proteins were fractioned according to their sub-cellular compartment. Protein extracts were immunoblotted, as indicated. i 3T3 cells were treated with 10 μM misoprostol or vehicle control for 4 h. Protein extracts were immunoblotted as indicated. j HCT-116 cells were transfected with wild-type P65 or empty vector control. Extracts were immunoblotted, as indicated. k HCT-116 cells were transfected with P65 constructs for 20 h. Extracts were immunoblotted, as indicated. Data are represented as mean ± S.E.M. * P

    Article Snippet: For H89 treatment, a 10 mM stock of H89-dihydrochloride hydrate (Sigma) in PBS, was diluted to 10 μM directly in media.

    Techniques: Expressing, Transfection, Fluorescence, Microscopy, Staining, Plasmid Preparation, Construct

    Effect of PKA inhibitor on BeWo cell fusion and expression profile of P-CREB and P-β-catenin (Ser675). BeWo cells were treated with H89 (10 μM) 1 h prior to forskolin (25 μM) treatment for varying time points using appropriate controls. Panel ( a ) represents fusion analysis carried out by desmoplakin staining at 48 h. Data is represented as fold change in fusion as compared with untreated control. Values are expressed as mean ± s.e.m. of three independent experiments. Representative desmoplakin I + II staining profile has also been shown. Scale bar represents 20 μm. Panel ( b ) shows representative Western blot and densitometric plot wherein band intensities normalized with their respective non-phosphorylated protein. The cropped blots were run under the same experimental conditions and the full length blots can be viewed in Supplementary Fig. S15 online . The data is expressed as fold change with respect to 0 h control and represents mean ± s.e.m. of three independent experiments. Representative blots for the same are also shown. *p

    Journal: Scientific Reports

    Article Title: Alpha or beta human chorionic gonadotropin knockdown decrease BeWo cell fusion by down-regulating PKA and CREB activation

    doi: 10.1038/srep11210

    Figure Lengend Snippet: Effect of PKA inhibitor on BeWo cell fusion and expression profile of P-CREB and P-β-catenin (Ser675). BeWo cells were treated with H89 (10 μM) 1 h prior to forskolin (25 μM) treatment for varying time points using appropriate controls. Panel ( a ) represents fusion analysis carried out by desmoplakin staining at 48 h. Data is represented as fold change in fusion as compared with untreated control. Values are expressed as mean ± s.e.m. of three independent experiments. Representative desmoplakin I + II staining profile has also been shown. Scale bar represents 20 μm. Panel ( b ) shows representative Western blot and densitometric plot wherein band intensities normalized with their respective non-phosphorylated protein. The cropped blots were run under the same experimental conditions and the full length blots can be viewed in Supplementary Fig. S15 online . The data is expressed as fold change with respect to 0 h control and represents mean ± s.e.m. of three independent experiments. Representative blots for the same are also shown. *p

    Article Snippet: Further, the cells were starved of FBS for at least 4 h before treatment with a pharmacological inhibitor of PKA, H89 dihydrochloride (10 μM; Sigma-Aldrich Inc.) for 1 h at 37 °C with 5% CO2 in humidified air.

    Techniques: Expressing, Staining, Western Blot

    Inhibition of PKA affected transcription and histone H4 acetylation near the transcription start sites of both Nurr1 and RANKL. (A,B) qPCR analysis of the mRNA level (A) and hnRNA level (B) of Nurr1 (left) and RANKL (right) in POBs pre-treated with PKA inhibitor H89 15 minutes prior to PTH treatment. hnRNA were determined by qPCR using primer sets amplifying exon-intron junction. Note that the maximum mRNA and hnRNA levels of both Nurr1 and RANKL were significantly down-regulated by H89 pre-treatment. Values were normalized by GAPDH and presented as a percentage of the maximum expression level (n = 3, mean±SEM, *p

    Journal: PLoS ONE

    Article Title: Different duration of parathyroid hormone exposure distinctively regulates primary response genes Nurr1 and RANKL in osteoblasts

    doi: 10.1371/journal.pone.0208514

    Figure Lengend Snippet: Inhibition of PKA affected transcription and histone H4 acetylation near the transcription start sites of both Nurr1 and RANKL. (A,B) qPCR analysis of the mRNA level (A) and hnRNA level (B) of Nurr1 (left) and RANKL (right) in POBs pre-treated with PKA inhibitor H89 15 minutes prior to PTH treatment. hnRNA were determined by qPCR using primer sets amplifying exon-intron junction. Note that the maximum mRNA and hnRNA levels of both Nurr1 and RANKL were significantly down-regulated by H89 pre-treatment. Values were normalized by GAPDH and presented as a percentage of the maximum expression level (n = 3, mean±SEM, *p

    Article Snippet: Prior to the experiments, cells were serum-starved overnight by changing medium to 1% FBS and 1% antibiotics, then treated with 10nM bovine PTH (1–34) (Sigma-Aldrich, St. Luis, MO) and/or 30 μM PKA inhibitor H89 (Sigma-Aldrich, #B1427) or 10μM MEK/ERK inhibitor U0126 (Sigma-Aldrich, #V1121) or 2 μM actin polymerization inhibitor Cytochalasin D (Calbiochem, #250255) or 2 μM Rho kinase (ROCK) inhibitor Y-27632 (Calbiochem, #688000) 15minutes prior to PTH for pre-treatment or 1 or 2 hours after PTH for post-treatment.

    Techniques: Inhibition, Real-time Polymerase Chain Reaction, Expressing

    Deletion of PfPDEβ bypasses the need for PKG activity upstream of MyoA S 19 phosphorylation in mature schizonts and results in premature proteolytic shedding of AMA1. (A) PKG-independent phosphorylation of MyoA in PfPDEβ-null parasites. Western blot of PKG inhibitor–treated (+C2) and E64-treated PfPDEβ ΔcatHA schizont proteins from DMSO- and RAP-treated PfPDEβ ΔcatHA cultures, probed with a phospho-S 19 MyoA antibody. The blot was reprobed with a polyclonal anti-MyoA antibody to determine the relative amounts of MyoA present in each sample. The lower panel shows total protein levels in the gel prior to blotting. The schematic shows the effect of various compounds on egress: Compound 2 and BAPTA-AM block egress upstream of PVM rupture, BIPPO induces egress, and E64 prevents erythrocyte plasma membrane rupture. (B) The PKA inhibitor H89 inhibits MyoA S 19 phosphorylation. Western blot analysis of E64-arrested wild-type schizonts (mock-treated PfPDEβ ΔcatHA ) treated with increasing concentrations of the PKA inhibitor H89. A sample was taken before inhibitor addition (start) and a Compound 2–blocked sample served as negative control. Blots were probed with antibodies against phospho-S 19 MyoA, total MyoA, and GAPDH as a loading control. (C) The PDE inhibitor BIPPO induces MyoA phosphorylation. Western blot analysis of Compound 2–arrested mock- or RAP-treated PfPDEβ ΔcatHA schizonts, following incubation with increasing concentrations of the PDE inhibitor BIPPO. A sample was taken before inhibitor addition to show the absence of MyoA S 19 phosphorylation (start). Blots were probed with the same antibodies as in (B). (D) BAPTA-AM inhibits MyoA phosphorylation. Effects of BAPTA-AM on MyoA S 19 phosphorylation in Compound 2–arrested (top panel) or E64-arrested (bottom panel) PfPDEβ ΔcatHA schizonts following mock or RAP treatment. Western blots were additionally probed with the same antibodies as in B and C. (E) Dual-stained IFAs showing the cellular distribution of AMA1 in RAP- and mock-treated (DMSO) PfPDEβ ΔcatHA schizonts. Schizonts were co-stained with an AMA1 antibody (red) and a HA antibody (green) to detect the presence or absence of PfPDEβ. DAPI (blue) was used to visualise nuclear material. Scale bar, 5 μm. The bar chart (right) shows quantitation of proportions of schizonts displaying apical or peripheral AMA1 staining in E64-arrested PfPDEβ ΔcatHA schizonts. Data presented are from a representative experiment in which > 50 schizonts per condition were counted by two researchers. Error bars, 1 SD. (F) Western blot analysis of culture supernatants from RAP- and mock-treated PfPDEβ ΔcatHA purified schizonts sampled over 90 minutes of culture. The blot was probed with antibodies specific for the micronemal proteins AMA1 and EBA175, as well as SERA5, to measure schizont rupture. Positions of molecular weight markers are indicated (left). The blot is representative of at least three independent experiments. (G) Quantitation of AMA1 shedding by IFA in merozoites released from RAP- and mock-treated PfPDEβ ΔcatHA schizonts. Purified merozoites were stained with an antibody against the AMA1 ectodomain. Thumbnails show examples for translocated (surface), shed (−), and micronemal (apical) AMA1 in green. Nuclei were stained with DAPI (blue). The bar charts show the mean proportions of merozoites exhibiting surface (left) and apical (right) AMA1 staining for each condition. More than 100 merozoites were counted by two researchers. Error bars, 1 SD. *, significant by unpaired t test ( p -value = 0.005); n.s., not significant ( p -value = 0.306). AMA1, apical membrane antigen-1; BAPTA-AM, 1,2-bis( o -aminophenoxy)ethane- N , N , N′ , N′ -tetraacetic acid-acetoxymethyl ester; BIPPO, 5-Benzyl-3-isopropyl-1 H -pyrazolo[4,3- d ]pyrimidin-7(6 H )-one; EBA175, erythrocyte-binding antigen 175; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HA, haemagglutinin; IFA, immunofluorescence assay; MyoA, myosin A; n.s., not significant; PDE, phosphodiesterase; PfPDEβ, Plasmodium falciparum phosphodiesterase β; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PVM, parasitophorous vacuole membrane; RAP, rapamycin; SERA5, serine repeat antigen 5.

    Journal: PLoS Biology

    Article Title: Phosphodiesterase beta is the master regulator of cAMP signalling during malaria parasite invasion

    doi: 10.1371/journal.pbio.3000154

    Figure Lengend Snippet: Deletion of PfPDEβ bypasses the need for PKG activity upstream of MyoA S 19 phosphorylation in mature schizonts and results in premature proteolytic shedding of AMA1. (A) PKG-independent phosphorylation of MyoA in PfPDEβ-null parasites. Western blot of PKG inhibitor–treated (+C2) and E64-treated PfPDEβ ΔcatHA schizont proteins from DMSO- and RAP-treated PfPDEβ ΔcatHA cultures, probed with a phospho-S 19 MyoA antibody. The blot was reprobed with a polyclonal anti-MyoA antibody to determine the relative amounts of MyoA present in each sample. The lower panel shows total protein levels in the gel prior to blotting. The schematic shows the effect of various compounds on egress: Compound 2 and BAPTA-AM block egress upstream of PVM rupture, BIPPO induces egress, and E64 prevents erythrocyte plasma membrane rupture. (B) The PKA inhibitor H89 inhibits MyoA S 19 phosphorylation. Western blot analysis of E64-arrested wild-type schizonts (mock-treated PfPDEβ ΔcatHA ) treated with increasing concentrations of the PKA inhibitor H89. A sample was taken before inhibitor addition (start) and a Compound 2–blocked sample served as negative control. Blots were probed with antibodies against phospho-S 19 MyoA, total MyoA, and GAPDH as a loading control. (C) The PDE inhibitor BIPPO induces MyoA phosphorylation. Western blot analysis of Compound 2–arrested mock- or RAP-treated PfPDEβ ΔcatHA schizonts, following incubation with increasing concentrations of the PDE inhibitor BIPPO. A sample was taken before inhibitor addition to show the absence of MyoA S 19 phosphorylation (start). Blots were probed with the same antibodies as in (B). (D) BAPTA-AM inhibits MyoA phosphorylation. Effects of BAPTA-AM on MyoA S 19 phosphorylation in Compound 2–arrested (top panel) or E64-arrested (bottom panel) PfPDEβ ΔcatHA schizonts following mock or RAP treatment. Western blots were additionally probed with the same antibodies as in B and C. (E) Dual-stained IFAs showing the cellular distribution of AMA1 in RAP- and mock-treated (DMSO) PfPDEβ ΔcatHA schizonts. Schizonts were co-stained with an AMA1 antibody (red) and a HA antibody (green) to detect the presence or absence of PfPDEβ. DAPI (blue) was used to visualise nuclear material. Scale bar, 5 μm. The bar chart (right) shows quantitation of proportions of schizonts displaying apical or peripheral AMA1 staining in E64-arrested PfPDEβ ΔcatHA schizonts. Data presented are from a representative experiment in which > 50 schizonts per condition were counted by two researchers. Error bars, 1 SD. (F) Western blot analysis of culture supernatants from RAP- and mock-treated PfPDEβ ΔcatHA purified schizonts sampled over 90 minutes of culture. The blot was probed with antibodies specific for the micronemal proteins AMA1 and EBA175, as well as SERA5, to measure schizont rupture. Positions of molecular weight markers are indicated (left). The blot is representative of at least three independent experiments. (G) Quantitation of AMA1 shedding by IFA in merozoites released from RAP- and mock-treated PfPDEβ ΔcatHA schizonts. Purified merozoites were stained with an antibody against the AMA1 ectodomain. Thumbnails show examples for translocated (surface), shed (−), and micronemal (apical) AMA1 in green. Nuclei were stained with DAPI (blue). The bar charts show the mean proportions of merozoites exhibiting surface (left) and apical (right) AMA1 staining for each condition. More than 100 merozoites were counted by two researchers. Error bars, 1 SD. *, significant by unpaired t test ( p -value = 0.005); n.s., not significant ( p -value = 0.306). AMA1, apical membrane antigen-1; BAPTA-AM, 1,2-bis( o -aminophenoxy)ethane- N , N , N′ , N′ -tetraacetic acid-acetoxymethyl ester; BIPPO, 5-Benzyl-3-isopropyl-1 H -pyrazolo[4,3- d ]pyrimidin-7(6 H )-one; EBA175, erythrocyte-binding antigen 175; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HA, haemagglutinin; IFA, immunofluorescence assay; MyoA, myosin A; n.s., not significant; PDE, phosphodiesterase; PfPDEβ, Plasmodium falciparum phosphodiesterase β; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PVM, parasitophorous vacuole membrane; RAP, rapamycin; SERA5, serine repeat antigen 5.

    Article Snippet: Small molecules and antibodies WR99210 was a kind gift from Jacobus Pharmaceuticals (New Jersey), RAP and the cysteine protease inhibitor E64 were purchased from Sigma, the PKG inhibitor Compound 2 was synthesised by MRC Technology (London, United Kingdom), the PKA inhibitor H89 was obtained from TOCRIS Biosciences, the PDE inhibitor BIPPO [ ] was a kind gift from Philip E. Thompson (Monash University, Australia).

    Techniques: Activity Assay, Western Blot, Blocking Assay, Negative Control, Incubation, Staining, Quantitation Assay, Purification, Molecular Weight, Immunofluorescence, Binding Assay

    Prolonged PKA activity in newly invaded PfPDEβ-null parasites blocks ring stage development. (A) The PDE inhibitor BIPPO phenocopies the PfPDEβ-null post-invasion phenotype. Giemsa-stained blood films showing the morphology of PfPDEβ ΔcatHA parasites at different time points in cycle 1, following either mock or RAP treatment of the parasites in cycle 0, compared to mock-treated parasites exposed to the PDE inhibitor BIPPO immediately following invasion. (B) The PKA inhibitor H89, but not the PKG inhibitor Compound 2, rescues BIPPO-treated early ring stages. The plots were generated by microscopic analysis of Giemsa-stained smears from ring stage cultures (18–22 hpi) treated with the indicated compounds. Kinase inhibitors (H89 and C2) were added at 1–5 hpi and BIPPO at 2–6 hpi. Scale bar, 5 μm. Data presented are mean counts (evaluated by three independent researchers) from two independent experiments, with > 100 parasites counted per condition. Error bars, SEM. *, significant by unpaired t test ( p -value = 0.0005); n.s., not significant ( p -value = 0.8508). (C) The PDE inhibitor BIPPO induces PKA-dependent phosphorylation in ring stages. Western blot of total protein from ring stages (12–16 hpi) treated for 90 minutes with various inhibitors: BIPPO (2 μM), H89 (30 μM), C2 (2 μM). Lane 1 = no inhibitor control, lane 2 = BIPPO only, lane 3 = BIPPO + H89, lane 4 = BIPPO + C2. The blot was probed with an antibody to phosphorylated PKA substrate motif. The gel was stained for total protein prior to blotting to show equal loading (right panel). BIPPO, 5-Benzyl-3-isopropyl-1 H -pyrazolo[4,3- d ]pyrimidin-7(6 H )-one; hpi, hours post invasion; PDE, phosphodiesterase; PfPDEβ, Plasmodium falciparum phosphodiesterase β; PKA, cAMP-dependent protein kinase; pS/pT, phosphoserine or phosphothreonine; RAP, rapamycin; R/K, arginine or lysine.

    Journal: PLoS Biology

    Article Title: Phosphodiesterase beta is the master regulator of cAMP signalling during malaria parasite invasion

    doi: 10.1371/journal.pbio.3000154

    Figure Lengend Snippet: Prolonged PKA activity in newly invaded PfPDEβ-null parasites blocks ring stage development. (A) The PDE inhibitor BIPPO phenocopies the PfPDEβ-null post-invasion phenotype. Giemsa-stained blood films showing the morphology of PfPDEβ ΔcatHA parasites at different time points in cycle 1, following either mock or RAP treatment of the parasites in cycle 0, compared to mock-treated parasites exposed to the PDE inhibitor BIPPO immediately following invasion. (B) The PKA inhibitor H89, but not the PKG inhibitor Compound 2, rescues BIPPO-treated early ring stages. The plots were generated by microscopic analysis of Giemsa-stained smears from ring stage cultures (18–22 hpi) treated with the indicated compounds. Kinase inhibitors (H89 and C2) were added at 1–5 hpi and BIPPO at 2–6 hpi. Scale bar, 5 μm. Data presented are mean counts (evaluated by three independent researchers) from two independent experiments, with > 100 parasites counted per condition. Error bars, SEM. *, significant by unpaired t test ( p -value = 0.0005); n.s., not significant ( p -value = 0.8508). (C) The PDE inhibitor BIPPO induces PKA-dependent phosphorylation in ring stages. Western blot of total protein from ring stages (12–16 hpi) treated for 90 minutes with various inhibitors: BIPPO (2 μM), H89 (30 μM), C2 (2 μM). Lane 1 = no inhibitor control, lane 2 = BIPPO only, lane 3 = BIPPO + H89, lane 4 = BIPPO + C2. The blot was probed with an antibody to phosphorylated PKA substrate motif. The gel was stained for total protein prior to blotting to show equal loading (right panel). BIPPO, 5-Benzyl-3-isopropyl-1 H -pyrazolo[4,3- d ]pyrimidin-7(6 H )-one; hpi, hours post invasion; PDE, phosphodiesterase; PfPDEβ, Plasmodium falciparum phosphodiesterase β; PKA, cAMP-dependent protein kinase; pS/pT, phosphoserine or phosphothreonine; RAP, rapamycin; R/K, arginine or lysine.

    Article Snippet: Small molecules and antibodies WR99210 was a kind gift from Jacobus Pharmaceuticals (New Jersey), RAP and the cysteine protease inhibitor E64 were purchased from Sigma, the PKG inhibitor Compound 2 was synthesised by MRC Technology (London, United Kingdom), the PKA inhibitor H89 was obtained from TOCRIS Biosciences, the PDE inhibitor BIPPO [ ] was a kind gift from Philip E. Thompson (Monash University, Australia).

    Techniques: Activity Assay, Staining, Generated, Western Blot

    YAP stabilizes phosphorylated CREB upon RA treatment. ( A ) Endogenous CREB was immunoprecipitated from WT and YAP KO cells after treatment with MG132 to block degradation and probed for ubiquitination (Ub). ( B ) Western blotting of CREB in cells treated with RA and H89. ( C ) Western blotting of p‐CREB and CREB after transiently expressed YAP in knockout cells. ( D ) Quantification of the percentage of neurite‐bearing cells and average neurite length in KO cells after transfected with YAP or empty vector. Data show the Mean ± S.E.M. values of three replicates (* P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: cAMP response element‐binding protein and Yes‐associated protein form a feedback loop that promotes neurite outgrowth

    doi: 10.1111/jcmm.13324

    Figure Lengend Snippet: YAP stabilizes phosphorylated CREB upon RA treatment. ( A ) Endogenous CREB was immunoprecipitated from WT and YAP KO cells after treatment with MG132 to block degradation and probed for ubiquitination (Ub). ( B ) Western blotting of CREB in cells treated with RA and H89. ( C ) Western blotting of p‐CREB and CREB after transiently expressed YAP in knockout cells. ( D ) Quantification of the percentage of neurite‐bearing cells and average neurite length in KO cells after transfected with YAP or empty vector. Data show the Mean ± S.E.M. values of three replicates (* P

    Article Snippet: To inhibit CREB activity, cells were treated with 20 μM H89 (Cayman Chemical Company, Ann Arbor, MI, USA).

    Techniques: Immunoprecipitation, Blocking Assay, Western Blot, Knock-Out, Transfection, Plasmid Preparation

    cAMP response element‐binding (CREB) promotes Yes‐associated protein (YAP) expression upon RA‐induced neurite outgrowth. ( A ) YAP gene and the CREB binding site are shown in UCSC genome browser. ( B ) Representative data of RA‐induced neurite outgrowth in N2a cells. ( C ) Western blotting of p‐CREB, total CREB, YAP and tubulin in cells treated with DMSO (Mock) or 5 μM RA for 24 hrs. ( D ) ChIP assay of CREB binding at YAP promoter or exon2 and intron2. ( E ) Western blotting of YAP expression in N2a cells treated with 20 μM CREB inhibitor H89.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: cAMP response element‐binding protein and Yes‐associated protein form a feedback loop that promotes neurite outgrowth

    doi: 10.1111/jcmm.13324

    Figure Lengend Snippet: cAMP response element‐binding (CREB) promotes Yes‐associated protein (YAP) expression upon RA‐induced neurite outgrowth. ( A ) YAP gene and the CREB binding site are shown in UCSC genome browser. ( B ) Representative data of RA‐induced neurite outgrowth in N2a cells. ( C ) Western blotting of p‐CREB, total CREB, YAP and tubulin in cells treated with DMSO (Mock) or 5 μM RA for 24 hrs. ( D ) ChIP assay of CREB binding at YAP promoter or exon2 and intron2. ( E ) Western blotting of YAP expression in N2a cells treated with 20 μM CREB inhibitor H89.

    Article Snippet: To inhibit CREB activity, cells were treated with 20 μM H89 (Cayman Chemical Company, Ann Arbor, MI, USA).

    Techniques: Binding Assay, Expressing, Western Blot, Chromatin Immunoprecipitation

    Blockade of Protein Kinase A impairs T3's effect on β-catenin, Cyclin-D1 and hepatocyte proliferation in mice A. A representative western blot using pooled samples from three wells per condition (left) shows increased levels of pSer675-β-catenin and pSer133-CREB in primary mouse hepatocytes after 30 minutes of T3 treatment. Inclusion of PKA inhibitor H89 (1 μM) in the media 30 minutes prior to the addition of T3 (100 nM) showed a notable decrease in pSer675-β-catenin and pSer133-CREB levels. Densitometry on the representative WB (right) shows an increase in pSer675-β-catenin and pSer133-CREB after T3 treatment, which was blocked by H89 treatment. (I.O.D. – integrated optical density). B. A representative western blot shows a decrease in the hepatic levels of pSer675-β-catenin and Cyclin-D1 when H89 was injected twice IP in 3-day T3 fed mice as compared to 3 day T3 only group. Gapdh verifies equal loading. Each lane represents an individual sample. C. A representative micrograph (200×) illustrates a decrease in the number of Cyclin-D1-positive hepatocytes when H89 was injected twice to the 3-day T3-fed mice as compared to T3 only group. Three or more mice per group were used for this study. D. Quantification of the Cyclin-D1-positive hepatocytes shows a significant decrease in positive cells in H89+T3 as compared to T3 only group (*p

    Journal: Hepatology (Baltimore, Md.)

    Article Title: Triiodothyronine induces hepatocyte proliferation by Protein Kinase A-Dependent β-Catenin Activation in Rodents

    doi: 10.1002/hep.26775

    Figure Lengend Snippet: Blockade of Protein Kinase A impairs T3's effect on β-catenin, Cyclin-D1 and hepatocyte proliferation in mice A. A representative western blot using pooled samples from three wells per condition (left) shows increased levels of pSer675-β-catenin and pSer133-CREB in primary mouse hepatocytes after 30 minutes of T3 treatment. Inclusion of PKA inhibitor H89 (1 μM) in the media 30 minutes prior to the addition of T3 (100 nM) showed a notable decrease in pSer675-β-catenin and pSer133-CREB levels. Densitometry on the representative WB (right) shows an increase in pSer675-β-catenin and pSer133-CREB after T3 treatment, which was blocked by H89 treatment. (I.O.D. – integrated optical density). B. A representative western blot shows a decrease in the hepatic levels of pSer675-β-catenin and Cyclin-D1 when H89 was injected twice IP in 3-day T3 fed mice as compared to 3 day T3 only group. Gapdh verifies equal loading. Each lane represents an individual sample. C. A representative micrograph (200×) illustrates a decrease in the number of Cyclin-D1-positive hepatocytes when H89 was injected twice to the 3-day T3-fed mice as compared to T3 only group. Three or more mice per group were used for this study. D. Quantification of the Cyclin-D1-positive hepatocytes shows a significant decrease in positive cells in H89+T3 as compared to T3 only group (*p

    Article Snippet: This was essentially similar to Experimental Protocol 1 except that H89 (200μg/100g/bw, LC Lab, Boston, MA) was injected IP every 24 hours for 5 days.

    Techniques: Mouse Assay, Western Blot, Injection

    Blockade of Protein Kinase A impairs T3's effect on β-catenin, Cyclin-D1 and hepatocyte proliferation in rats A. Representative microphotographs (200×) illustrate the effect of H89 on T3-induced rat hepatocyte proliferation by BrdU immunohistochemistry. H89 was given 1 hour prior to a single dose of T3 (20 μg/100 g) and the rats were sacrificed 24 hours later. Minimum four rats per group were used for this entire study. B. Quantification of BrdU positive hepatocytes in A shows a significant (*p

    Journal: Hepatology (Baltimore, Md.)

    Article Title: Triiodothyronine induces hepatocyte proliferation by Protein Kinase A-Dependent β-Catenin Activation in Rodents

    doi: 10.1002/hep.26775

    Figure Lengend Snippet: Blockade of Protein Kinase A impairs T3's effect on β-catenin, Cyclin-D1 and hepatocyte proliferation in rats A. Representative microphotographs (200×) illustrate the effect of H89 on T3-induced rat hepatocyte proliferation by BrdU immunohistochemistry. H89 was given 1 hour prior to a single dose of T3 (20 μg/100 g) and the rats were sacrificed 24 hours later. Minimum four rats per group were used for this entire study. B. Quantification of BrdU positive hepatocytes in A shows a significant (*p

    Article Snippet: This was essentially similar to Experimental Protocol 1 except that H89 (200μg/100g/bw, LC Lab, Boston, MA) was injected IP every 24 hours for 5 days.

    Techniques: Immunohistochemistry

    Proliferative focus morphology. Sertoli cells were treated for 24 h with retinol in the presence or absence of the antioxidant Trolox (100 μmol/L), the p38 inhibitor SB203580 (10 μmol/L), the JNK inhibitor SP600125 (10 μmol/L), the Akt inhibitor LY294002 (10 μmol/L), the ERK inhibitor U0126 (10 μmol/L) the pan-PKC inhibitor GÖ6983 (10 μmol/L) and the PKA inhibitor H89 (1 μmol/L); medium was then replaced in all groups for 10% FBS-supplemented 199 medium without any other compound for 14 d, with exchange for fresh medium every 3 d. At the end of the 14 d period, morphology was examined in a phase-contrast microscope (representative micrographs at ×40 are depicted) and proliferative foci were counted in each well (see Figure 5A for scores).

    Journal: Acta Pharmacologica Sinica

    Article Title: Retinol induces morphological alterations and proliferative focus formation through free radical-mediated activation of multiple signaling pathways

    doi: 10.1038/aps.2011.202

    Figure Lengend Snippet: Proliferative focus morphology. Sertoli cells were treated for 24 h with retinol in the presence or absence of the antioxidant Trolox (100 μmol/L), the p38 inhibitor SB203580 (10 μmol/L), the JNK inhibitor SP600125 (10 μmol/L), the Akt inhibitor LY294002 (10 μmol/L), the ERK inhibitor U0126 (10 μmol/L) the pan-PKC inhibitor GÖ6983 (10 μmol/L) and the PKA inhibitor H89 (1 μmol/L); medium was then replaced in all groups for 10% FBS-supplemented 199 medium without any other compound for 14 d, with exchange for fresh medium every 3 d. At the end of the 14 d period, morphology was examined in a phase-contrast microscope (representative micrographs at ×40 are depicted) and proliferative foci were counted in each well (see Figure 5A for scores).

    Article Snippet: U0126 was from Promega Corporation (Madison, WI, USA), GÖ6983 and SB203580 were from Merck Biosciences (Darmstadt, Germany) and H89 was from Biomol Research Laboratories (Plymouth Meeting, PA, USA).

    Techniques: Microscopy

    Proliferative focus scores and [ 3 H]dT incorporation in Sertoli cells. (A) Sertoli cells were treated for 24 h with retinol in the presence or absence of the antioxidant Trolox (100 μmol/L), the p38 inhibitor SB203580 (10 μmol/L), the JNK inhibitor SP600125 (10 μmol/L), the Akt inhibitor LY294002 (10 μmol/L), the ERK inhibitor U0126 (10 μmol/L) the pan-PKC inhibitor GÖ6983 (10 μmol/L) and the PKA inhibitor H89 (1 μmol/L); medium was then replaced in all groups for 10% FBS-supplemented 199 medium without any other compound for 14 d, with exchange for fresh medium every 3 d. At the end of the 14 d period, proliferative foci were counted in each well. (B) Sertoli cells were previously incubated with medium containing [ 3 H]dT, and then treated for 24 h in the same medium with retinol 7 μmol/L in the presence or absence of the above-mentioned compounds. Nuclei were isolated and incorporation of [ 3 H]dT was counted. Bars represent mean±SEM from three independent experiments (triplicate); data were analyzed by one-way ANOVA with Duncan's post hoc test. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Retinol induces morphological alterations and proliferative focus formation through free radical-mediated activation of multiple signaling pathways

    doi: 10.1038/aps.2011.202

    Figure Lengend Snippet: Proliferative focus scores and [ 3 H]dT incorporation in Sertoli cells. (A) Sertoli cells were treated for 24 h with retinol in the presence or absence of the antioxidant Trolox (100 μmol/L), the p38 inhibitor SB203580 (10 μmol/L), the JNK inhibitor SP600125 (10 μmol/L), the Akt inhibitor LY294002 (10 μmol/L), the ERK inhibitor U0126 (10 μmol/L) the pan-PKC inhibitor GÖ6983 (10 μmol/L) and the PKA inhibitor H89 (1 μmol/L); medium was then replaced in all groups for 10% FBS-supplemented 199 medium without any other compound for 14 d, with exchange for fresh medium every 3 d. At the end of the 14 d period, proliferative foci were counted in each well. (B) Sertoli cells were previously incubated with medium containing [ 3 H]dT, and then treated for 24 h in the same medium with retinol 7 μmol/L in the presence or absence of the above-mentioned compounds. Nuclei were isolated and incorporation of [ 3 H]dT was counted. Bars represent mean±SEM from three independent experiments (triplicate); data were analyzed by one-way ANOVA with Duncan's post hoc test. b P

    Article Snippet: U0126 was from Promega Corporation (Madison, WI, USA), GÖ6983 and SB203580 were from Merck Biosciences (Darmstadt, Germany) and H89 was from Biomol Research Laboratories (Plymouth Meeting, PA, USA).

    Techniques: Incubation, Isolation

    Effect of different protein kinase inhibitors on cell morphology modification by retinol. (A) Sertoli cells were treated with retinol 7 μmol/L in the presence or absence of the antioxidant Trolox (100 μmol/L), the p38 inhibitor SB203580 (10 μmol/L), the JNK inhibitor SP600125 (10 μmol/L), the Akt inhibitor LY294002 (10 μmol/L), the ERK inhibitor U0126 (10 μmol/L) the pan-PKC inhibitor GÖ6983 (10 μmol/L) and the PKA inhibitor H89 (1 μmol/L) for 24 h, and the cell morphology was examined by phase-contrast microscopy (Nikon Eclipse TE 300, ×100). (B) Cell deformation after 24 h was also analyzed by calculation of the coefficient D , an index of cell transformation-related deformation, obtained by morphometrical measurement of scanned phase-contrast photomicrographs of cells plated at dispersed and confluent densities. Bars represent mean±SEM from 22 individual cells analyzed per each group; data were analyzed by one-way ANOVA with Duncan's post hoc test. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Retinol induces morphological alterations and proliferative focus formation through free radical-mediated activation of multiple signaling pathways

    doi: 10.1038/aps.2011.202

    Figure Lengend Snippet: Effect of different protein kinase inhibitors on cell morphology modification by retinol. (A) Sertoli cells were treated with retinol 7 μmol/L in the presence or absence of the antioxidant Trolox (100 μmol/L), the p38 inhibitor SB203580 (10 μmol/L), the JNK inhibitor SP600125 (10 μmol/L), the Akt inhibitor LY294002 (10 μmol/L), the ERK inhibitor U0126 (10 μmol/L) the pan-PKC inhibitor GÖ6983 (10 μmol/L) and the PKA inhibitor H89 (1 μmol/L) for 24 h, and the cell morphology was examined by phase-contrast microscopy (Nikon Eclipse TE 300, ×100). (B) Cell deformation after 24 h was also analyzed by calculation of the coefficient D , an index of cell transformation-related deformation, obtained by morphometrical measurement of scanned phase-contrast photomicrographs of cells plated at dispersed and confluent densities. Bars represent mean±SEM from 22 individual cells analyzed per each group; data were analyzed by one-way ANOVA with Duncan's post hoc test. b P

    Article Snippet: U0126 was from Promega Corporation (Madison, WI, USA), GÖ6983 and SB203580 were from Merck Biosciences (Darmstadt, Germany) and H89 was from Biomol Research Laboratories (Plymouth Meeting, PA, USA).

    Techniques: Modification, Microscopy, Transformation Assay

    Phosphorylation at S675 in β-catenin E654 by protein kinase A (PKA). (A–C) Immunoblotting showing total β-catenin (βcat) and phosphoS675 β -catenin (pS675) expression levels in total lysates of wild-type (YY), β-catenin Y654/E654 (YE) and β-catenin E654/E654 (EE) mouse embryonic fibroblasts (MEF) (A), embryos (B) and intestinal tumours from Apc and Apc;YE mice (C). Tubulin (TUB) expression levels were used as a loading control. The relative expression levels were determined in three individual experiments and averaged. Numbers between brackets refer to the number of independent cell lines used for each genotype. (D, E) MEF were treated with H89 (D) or forskolin (FSK) (E). Effects on PKA activity were evaluated by determining the expression levels of phosphoCREB (pCREB, upper band; lower band is phosphoATF-1). Following treatment, phosphoS675 β -catenin expression levels were determined and quantified. DMSO, dimethyl sulphoxide.

    Journal: Gut

    Article Title: β-catenin tyrosine 654 phosphorylation increases Wnt signalling and intestinal tumorigenesis

    doi: 10.1136/gut.2010.233460

    Figure Lengend Snippet: Phosphorylation at S675 in β-catenin E654 by protein kinase A (PKA). (A–C) Immunoblotting showing total β-catenin (βcat) and phosphoS675 β -catenin (pS675) expression levels in total lysates of wild-type (YY), β-catenin Y654/E654 (YE) and β-catenin E654/E654 (EE) mouse embryonic fibroblasts (MEF) (A), embryos (B) and intestinal tumours from Apc and Apc;YE mice (C). Tubulin (TUB) expression levels were used as a loading control. The relative expression levels were determined in three individual experiments and averaged. Numbers between brackets refer to the number of independent cell lines used for each genotype. (D, E) MEF were treated with H89 (D) or forskolin (FSK) (E). Effects on PKA activity were evaluated by determining the expression levels of phosphoCREB (pCREB, upper band; lower band is phosphoATF-1). Following treatment, phosphoS675 β -catenin expression levels were determined and quantified. DMSO, dimethyl sulphoxide.

    Article Snippet: Cells were treated with 25 μM H89 (Enzo Life Sciences, Lausen, Switzerland) for 24 h or 50 μM forskolin (Merck, Whitehouse Station, NJ, USA) for 1 h.

    Techniques: Expressing, Mouse Assay, Activity Assay

    The traces of the 60 s spatial probe trials, the frequency of swimming across the platform site, and the swimming speed of the five groups. Cerebral hypoperfusion effected the spatial probe trial as demonstrated by the decreased frequency of swimming across the platform during the 60 s interval. In the EA group, the frequency increased (a); after ICV injection of H89, the increased frequency decreased (b), while there were no significant differences in the swimming speed either among the control, model, and EA groups or between the EA + NS and EA + H89 groups ((c) and (d)). The data are expressed as the mean ± SEM. ANOVA statistical analyses were performed to compare the means among the control, model, and EA groups; ※ P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Electroacupuncture Ameliorates Learning and Memory via Activation of the CREB Signaling Pathway in the Hippocampus to Attenuate Apoptosis after Cerebral Hypoperfusion

    doi: 10.1155/2013/156489

    Figure Lengend Snippet: The traces of the 60 s spatial probe trials, the frequency of swimming across the platform site, and the swimming speed of the five groups. Cerebral hypoperfusion effected the spatial probe trial as demonstrated by the decreased frequency of swimming across the platform during the 60 s interval. In the EA group, the frequency increased (a); after ICV injection of H89, the increased frequency decreased (b), while there were no significant differences in the swimming speed either among the control, model, and EA groups or between the EA + NS and EA + H89 groups ((c) and (d)). The data are expressed as the mean ± SEM. ANOVA statistical analyses were performed to compare the means among the control, model, and EA groups; ※ P

    Article Snippet: H89 (2 μ g/μ L × 10 μ L, Beyotime Institute of Biotechnology, Shanghai, China.) was given intracerebroventricularly in the right hemisphere, 5–10 min before 2VO, similar to a previous study [ ].

    Techniques: Injection

    Western blotting showing immunoreactivity of phosphorylated CREB in the hippocampal CA1 area. The pCREB protein values were calculated as a ratio of pCREB protein to GAPDH. EA increased the expression of pCREB protein (a), and the EA-induced activation of pCREB was markedly inhibited by ICV injection of H89 (b). The data are expressed as the mean ± SEM. ANOVA statistical analyses were performed to compare the means among the control, model, and EA groups; ※ P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Electroacupuncture Ameliorates Learning and Memory via Activation of the CREB Signaling Pathway in the Hippocampus to Attenuate Apoptosis after Cerebral Hypoperfusion

    doi: 10.1155/2013/156489

    Figure Lengend Snippet: Western blotting showing immunoreactivity of phosphorylated CREB in the hippocampal CA1 area. The pCREB protein values were calculated as a ratio of pCREB protein to GAPDH. EA increased the expression of pCREB protein (a), and the EA-induced activation of pCREB was markedly inhibited by ICV injection of H89 (b). The data are expressed as the mean ± SEM. ANOVA statistical analyses were performed to compare the means among the control, model, and EA groups; ※ P

    Article Snippet: H89 (2 μ g/μ L × 10 μ L, Beyotime Institute of Biotechnology, Shanghai, China.) was given intracerebroventricularly in the right hemisphere, 5–10 min before 2VO, similar to a previous study [ ].

    Techniques: Western Blot, Expressing, Activation Assay, Injection

    Western blotting showing immunoreactivity of apoptosis-related proteins in the hippocampal CA1 area. The Bcl-2 and Bax protein values were calculated as a ratio of Bcl-2 and Bax protein to GAPDH. EA increased the expression of Bcl-2 and decreased the expression of Bax ((a) and (c)). However, the antiapoptotic action of EA was markedly inhibited by ICV injection of H89 ((b) and (d)). The data are expressed as the mean ± SEM. ANOVA statistical analyses were performed to compare the means among the control, model, and EA groups; △ P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Electroacupuncture Ameliorates Learning and Memory via Activation of the CREB Signaling Pathway in the Hippocampus to Attenuate Apoptosis after Cerebral Hypoperfusion

    doi: 10.1155/2013/156489

    Figure Lengend Snippet: Western blotting showing immunoreactivity of apoptosis-related proteins in the hippocampal CA1 area. The Bcl-2 and Bax protein values were calculated as a ratio of Bcl-2 and Bax protein to GAPDH. EA increased the expression of Bcl-2 and decreased the expression of Bax ((a) and (c)). However, the antiapoptotic action of EA was markedly inhibited by ICV injection of H89 ((b) and (d)). The data are expressed as the mean ± SEM. ANOVA statistical analyses were performed to compare the means among the control, model, and EA groups; △ P

    Article Snippet: H89 (2 μ g/μ L × 10 μ L, Beyotime Institute of Biotechnology, Shanghai, China.) was given intracerebroventricularly in the right hemisphere, 5–10 min before 2VO, similar to a previous study [ ].

    Techniques: Western Blot, Expressing, Injection

    The traces of swimming in place navigation trials, the latency time to find the submerged platform, and the swimming speed of the five groups. The grey lines are the swimming traces, which end at the platforms (the small red circles in quadrant III). EA decreased the latency time of rats with cerebral hypoperfusion (a), and after ICV injection of H89, rats demonstrated prolonged latency (b), while there were no significant differences in the swimming speed either among the control, model, and EA groups or between the EA + NS and EA + H89 groups ((c) and (d)). The data are expressed as the mean ± SEM. ANOVA statistical analyses were performed to compare the means among the control, model, and EA groups; △ P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Electroacupuncture Ameliorates Learning and Memory via Activation of the CREB Signaling Pathway in the Hippocampus to Attenuate Apoptosis after Cerebral Hypoperfusion

    doi: 10.1155/2013/156489

    Figure Lengend Snippet: The traces of swimming in place navigation trials, the latency time to find the submerged platform, and the swimming speed of the five groups. The grey lines are the swimming traces, which end at the platforms (the small red circles in quadrant III). EA decreased the latency time of rats with cerebral hypoperfusion (a), and after ICV injection of H89, rats demonstrated prolonged latency (b), while there were no significant differences in the swimming speed either among the control, model, and EA groups or between the EA + NS and EA + H89 groups ((c) and (d)). The data are expressed as the mean ± SEM. ANOVA statistical analyses were performed to compare the means among the control, model, and EA groups; △ P

    Article Snippet: H89 (2 μ g/μ L × 10 μ L, Beyotime Institute of Biotechnology, Shanghai, China.) was given intracerebroventricularly in the right hemisphere, 5–10 min before 2VO, similar to a previous study [ ].

    Techniques: Injection

    FCS and FCCS analyses in live cells expressing DsRed-AQP2 and GFP-actin. (A and D) The crosshair indicates the measurement position: A, subapical region; D, apical membrane. Images for GFP-actin (green) and DsRed-AQP2 (red) were overlayed. In all images, the orthogonal z -stack profiles along the indicated lines in the x or y dimension of the central xy image are shown in the rectangular windows at the bottom (xz) and to the right (yz) of the central image. Bars, 5 μm. (B and E) FCCS analysis in the subapical region (B) and the apical membrane (E). Autocorrelation curves at the positions indicated in the images above are shown. Blue lines correspond to GFP-actin signal, red lines correspond to DsRed-AQP2 signal, and black lines are cross-correlation curves. The dashed lines are measured curves and the solid lines are their fitted curves. Insets show the fluorescence intensities of DsRed-AQP2 and GFP-actin in the two respective channels during an FCCS measurement. CR, count rates. (C and F) Cross-correlation curves before (Before FK) and after (After FK) forskolin stimulation. (G) Summary of relative cross-correlation amplitude ([ Gc (0)-1]/[ Gr (0)-1]). C, control; FK, forskolin treatment; H89, H89-pretreated cells expressing DsRed-AQP2 and GFP-actin; S256A, cells expressing DsRed-S256A-AQP2 and GFP-actin; subapical, measurements in the subapical region; apical, measurements on the apical membrane. Data represent the mean and SE from at least five independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: Reciprocal interaction with G-actin and tropomyosin is essential for aquaporin-2 trafficking

    doi: 10.1083/jcb.200709177

    Figure Lengend Snippet: FCS and FCCS analyses in live cells expressing DsRed-AQP2 and GFP-actin. (A and D) The crosshair indicates the measurement position: A, subapical region; D, apical membrane. Images for GFP-actin (green) and DsRed-AQP2 (red) were overlayed. In all images, the orthogonal z -stack profiles along the indicated lines in the x or y dimension of the central xy image are shown in the rectangular windows at the bottom (xz) and to the right (yz) of the central image. Bars, 5 μm. (B and E) FCCS analysis in the subapical region (B) and the apical membrane (E). Autocorrelation curves at the positions indicated in the images above are shown. Blue lines correspond to GFP-actin signal, red lines correspond to DsRed-AQP2 signal, and black lines are cross-correlation curves. The dashed lines are measured curves and the solid lines are their fitted curves. Insets show the fluorescence intensities of DsRed-AQP2 and GFP-actin in the two respective channels during an FCCS measurement. CR, count rates. (C and F) Cross-correlation curves before (Before FK) and after (After FK) forskolin stimulation. (G) Summary of relative cross-correlation amplitude ([ Gc (0)-1]/[ Gr (0)-1]). C, control; FK, forskolin treatment; H89, H89-pretreated cells expressing DsRed-AQP2 and GFP-actin; S256A, cells expressing DsRed-S256A-AQP2 and GFP-actin; subapical, measurements in the subapical region; apical, measurements on the apical membrane. Data represent the mean and SE from at least five independent experiments. *, P

    Article Snippet: Cells were stimulated with 40 μM forskolin or preincubated with 30 μM H89 (Seikagaku Corporation) for 30 min before being treated with 40 μM forskolin for 10 min in the presence of 30 μM H89.

    Techniques: Expressing, Fluorescence

    TM5b knockdown promotes AQP2 trafficking in MDCK/AQP2 cells. (A–D) MDCK/AQP2 cells were transfected with TM5b siRNA (B) or scrambled control siRNA (A), which was indicated by fluorescein-labeled dsRNA (red), and treated without (top) or with (bottom) forskolin. Pretreatment with H89 was also performed using the cells transfected with TM5b siRNA (D) or the control siRNA (C). Cells were labeled for AQP2 (green). Bars, 10 μm. (E and F) Apical cell surface biotinylation assay using MDCK/AQP2 cells transfected with TM5b siRNA or control siRNA, corresponding to A–D. (E) Biotinylated proteins were precipitated with streptavidin-agarose beads and immunoblotted for AQP2. (F) The densitometric quantification normalized to control siRNA-transfected cells without forskolin treatment. Data represent the mean and SE from three independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: Reciprocal interaction with G-actin and tropomyosin is essential for aquaporin-2 trafficking

    doi: 10.1083/jcb.200709177

    Figure Lengend Snippet: TM5b knockdown promotes AQP2 trafficking in MDCK/AQP2 cells. (A–D) MDCK/AQP2 cells were transfected with TM5b siRNA (B) or scrambled control siRNA (A), which was indicated by fluorescein-labeled dsRNA (red), and treated without (top) or with (bottom) forskolin. Pretreatment with H89 was also performed using the cells transfected with TM5b siRNA (D) or the control siRNA (C). Cells were labeled for AQP2 (green). Bars, 10 μm. (E and F) Apical cell surface biotinylation assay using MDCK/AQP2 cells transfected with TM5b siRNA or control siRNA, corresponding to A–D. (E) Biotinylated proteins were precipitated with streptavidin-agarose beads and immunoblotted for AQP2. (F) The densitometric quantification normalized to control siRNA-transfected cells without forskolin treatment. Data represent the mean and SE from three independent experiments. *, P

    Article Snippet: Cells were stimulated with 40 μM forskolin or preincubated with 30 μM H89 (Seikagaku Corporation) for 30 min before being treated with 40 μM forskolin for 10 min in the presence of 30 μM H89.

    Techniques: Transfection, Labeling, Cell Surface Biotinylation Assay

    S. aureus lipoproteins and host TLR2, TLR4, and NLRP3 are involved in secretion of PGE 2 and proinflammatory cytokines and chemokines by macrophages after S. aureus infection. WT and corresponding gene-deficient macrophages were infected with multiple S. aureus strains (SA113, Δ lgt , and + pRB) at MOI 10:1 or not infected. PGE 2 , TNF-α, IL-1β, and RANTES release into the supernatant of macrophage cultures was analyzed by ELISA 9 h after infection ( a–h ). Activation of the cAMP-PKA (P-PKA) and MAPK (P-ERK and P-p38) pathways was evaluated by western blotting. GAPDH served as a loading control ( i , j ). WT macrophages (untreated or pre-treated with 10 µM H89 for 2 h) were infected with WT S. aureus strains at MOI 10:1 or not infected. TNF-α, IL-1β, and RANTES production in the supernatants of macrophages was analyzed by ELISA 9 h after infection ( k ). Results are expressed as the mean ± SD of 3 independent experiments and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test or 2-way ANOVA with Bonferroni's post-test. * p

    Journal: Journal of Innate Immunity

    Article Title: Prostaglandin E2 Regulates Activation of Mouse Peritoneal Macrophages by Staphylococcus aureus through Toll-Like Receptor 2, Toll-Like Receptor 4, and NLRP3 Inflammasome Signaling

    doi: 10.1159/000499604

    Figure Lengend Snippet: S. aureus lipoproteins and host TLR2, TLR4, and NLRP3 are involved in secretion of PGE 2 and proinflammatory cytokines and chemokines by macrophages after S. aureus infection. WT and corresponding gene-deficient macrophages were infected with multiple S. aureus strains (SA113, Δ lgt , and + pRB) at MOI 10:1 or not infected. PGE 2 , TNF-α, IL-1β, and RANTES release into the supernatant of macrophage cultures was analyzed by ELISA 9 h after infection ( a–h ). Activation of the cAMP-PKA (P-PKA) and MAPK (P-ERK and P-p38) pathways was evaluated by western blotting. GAPDH served as a loading control ( i , j ). WT macrophages (untreated or pre-treated with 10 µM H89 for 2 h) were infected with WT S. aureus strains at MOI 10:1 or not infected. TNF-α, IL-1β, and RANTES production in the supernatants of macrophages was analyzed by ELISA 9 h after infection ( k ). Results are expressed as the mean ± SD of 3 independent experiments and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test or 2-way ANOVA with Bonferroni's post-test. * p

    Article Snippet: In the groups treated with the PKA inhibitor H89 (Cayman Chemical Company, Ann Arbor, MI, USA), macrophages were incubated in culture media supplemented with 10 µM H89 for 2 h before infection.

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot

    YC-1 activates cyclic nucleotide-dependent protein kinases in A549 cells. ( A ) A549 cells were pretreated with YC-1 for 1 h and incubated under normoxia or hypoxia for another 1 h. Cell lysates were subjected to Western blotting for phospho-VASP. ( B , C ) A549 cells were pretreated with the PDE inhibitor IBMX (100 μM), the sGC inhibitor ODQ (10 μM), or the PKA inhibitor H89 (5 μM) for 30 min, then treated with YC-1 or BAY 41-2272 (BAY) for 1 h and incubated under hypoxia for another 24 h. TF expression was determined by Western blotting.

    Journal: International Journal of Molecular Sciences

    Article Title: YC-1 Prevents Tumor-Associated Tissue Factor Expression and Procoagulant Activity in Hypoxic Conditions by Inhibiting p38/NF-κB Signaling Pathway

    doi: 10.3390/ijms20020244

    Figure Lengend Snippet: YC-1 activates cyclic nucleotide-dependent protein kinases in A549 cells. ( A ) A549 cells were pretreated with YC-1 for 1 h and incubated under normoxia or hypoxia for another 1 h. Cell lysates were subjected to Western blotting for phospho-VASP. ( B , C ) A549 cells were pretreated with the PDE inhibitor IBMX (100 μM), the sGC inhibitor ODQ (10 μM), or the PKA inhibitor H89 (5 μM) for 30 min, then treated with YC-1 or BAY 41-2272 (BAY) for 1 h and incubated under hypoxia for another 24 h. TF expression was determined by Western blotting.

    Article Snippet: The PKA inhibitor H89 were purchased from Enzo Life Sciences (Farmingdale, NY, USA).

    Techniques: Incubation, Western Blot, Expressing

    The role of PKA and CK2 in regulating SK2 channels in intrinsic plasticity. A , Example traces for cells treated with H89, a PKA inhibitor that was present in the bath for the duration of the experiment. B , Time graph for changes in spiking relative to baseline. Either somatic depolarization or oxo-m was applied from minute 5 to 10. C , Example traces for cells treated with TBB, a CK2 inhibitor that was present in the bath for the duration of the experiment. D , Time graph for changes in spiking relative to baseline. Either somatic depolarization or oxo-m was applied from minute 5 to 10. E , Bar graph for depolarization-induced changes in spiking relative to baseline. Both H89 and TBB bath application prevented intrinsic plasticity. F , Bar graph for oxo-m-induced changes in spiking relative to baseline. H89, but not TBB, prevented intrinsic plasticity; * p

    Journal: eNeuro

    Article Title: Muscarinic Modulation of SK2-Type K+ Channels Promotes Intrinsic Plasticity in L2/3 Pyramidal Neurons of the Mouse Primary Somatosensory Cortex

    doi: 10.1523/ENEURO.0453-19.2020

    Figure Lengend Snippet: The role of PKA and CK2 in regulating SK2 channels in intrinsic plasticity. A , Example traces for cells treated with H89, a PKA inhibitor that was present in the bath for the duration of the experiment. B , Time graph for changes in spiking relative to baseline. Either somatic depolarization or oxo-m was applied from minute 5 to 10. C , Example traces for cells treated with TBB, a CK2 inhibitor that was present in the bath for the duration of the experiment. D , Time graph for changes in spiking relative to baseline. Either somatic depolarization or oxo-m was applied from minute 5 to 10. E , Bar graph for depolarization-induced changes in spiking relative to baseline. Both H89 and TBB bath application prevented intrinsic plasticity. F , Bar graph for oxo-m-induced changes in spiking relative to baseline. H89, but not TBB, prevented intrinsic plasticity; * p

    Article Snippet: H89 (PKA inhibitor) was purchased from Abcam.

    Techniques:

    Effect of the PKA inhibitor H-89 on agonist-induced ERK1/2 phosphorylation in D1R and D1R-D3R cells. ERK1/2 phosphorylation in CHO cells transfected with D1R-Rluc cDNA (1 μg cDNA) alone (A) or D1R-Rluc (1 μg cDNA) and D3R-YFP (1.3 μg cDNA) (B-D). 48 h post-transfection, D1R or D1R-D3R cells were pre-treated or not with the PKA inhibitor H-89 (10 μM for 30 min) and were treated for 7 min with the D1R agonist SKF 38393 (1 μM) (A-D), the D3R agonist PD 128907 (1 μM) (C-D) or both (C-D). Quantification of phosphorylated ERK1/2 was determined by Western blot. Representative blots are shown. Values are mean ± S.E.M. (n = 4 with duplicates) and are expressed as percentage of basal or non-treated cells; * , ** and ***: p

    Journal: Molecular neurobiology

    Article Title: Biased G Protein-Independent Signaling of Dopamine D1-D3 Receptor Heteromers in the Nucleus Accumbens

    doi: 10.1007/s12035-019-1564-8

    Figure Lengend Snippet: Effect of the PKA inhibitor H-89 on agonist-induced ERK1/2 phosphorylation in D1R and D1R-D3R cells. ERK1/2 phosphorylation in CHO cells transfected with D1R-Rluc cDNA (1 μg cDNA) alone (A) or D1R-Rluc (1 μg cDNA) and D3R-YFP (1.3 μg cDNA) (B-D). 48 h post-transfection, D1R or D1R-D3R cells were pre-treated or not with the PKA inhibitor H-89 (10 μM for 30 min) and were treated for 7 min with the D1R agonist SKF 38393 (1 μM) (A-D), the D3R agonist PD 128907 (1 μM) (C-D) or both (C-D). Quantification of phosphorylated ERK1/2 was determined by Western blot. Representative blots are shown. Values are mean ± S.E.M. (n = 4 with duplicates) and are expressed as percentage of basal or non-treated cells; * , ** and ***: p

    Article Snippet: Transfected CHO cells were cultured in serum-free medium for 16 h before the addition of any agent and were not treated or treated with the PKA inhibitor H-89 (10 μM; Tocris, Pittsburgh, PA) for 30 min at 37 °C.

    Techniques: Transfection, Western Blot

    Effect of the PKA inhibitor H-89 and D1R TAT-TM peptides on D1R-D3R agonist-induced striatal ERK1/2 phosphorylation in reserpinized mice. ) to process the brains for the immunohistochemical analysis of ERK1/2 phosphorylation. Positive cells were counted within the area of the shell of the NAc of both sides from several coronal sections immediate anterior to the cannula implantation (from 2.28 to 1.44 mm anterior to bregma) and, for each animal, values from the side ipsilateral to the intracerebral infusion was compared to the contralateral side. Values (n = 6–9) are expressed as number of cells/mm 2 . P values represent the results of paired t test, used to determine significant differences between the NAc ipsilateral to the intracranial infusion versus the contralateral side.

    Journal: Molecular neurobiology

    Article Title: Biased G Protein-Independent Signaling of Dopamine D1-D3 Receptor Heteromers in the Nucleus Accumbens

    doi: 10.1007/s12035-019-1564-8

    Figure Lengend Snippet: Effect of the PKA inhibitor H-89 and D1R TAT-TM peptides on D1R-D3R agonist-induced striatal ERK1/2 phosphorylation in reserpinized mice. ) to process the brains for the immunohistochemical analysis of ERK1/2 phosphorylation. Positive cells were counted within the area of the shell of the NAc of both sides from several coronal sections immediate anterior to the cannula implantation (from 2.28 to 1.44 mm anterior to bregma) and, for each animal, values from the side ipsilateral to the intracerebral infusion was compared to the contralateral side. Values (n = 6–9) are expressed as number of cells/mm 2 . P values represent the results of paired t test, used to determine significant differences between the NAc ipsilateral to the intracranial infusion versus the contralateral side.

    Article Snippet: Transfected CHO cells were cultured in serum-free medium for 16 h before the addition of any agent and were not treated or treated with the PKA inhibitor H-89 (10 μM; Tocris, Pittsburgh, PA) for 30 min at 37 °C.

    Techniques: Mouse Assay, Immunohistochemistry