Journal: Cell Cycle

Article Title: Binding of FGF2 to FGFR2 in an autocrine mode in trophectoderm cells is indispensable for mouse blastocyst formation through PKC-p38 pathway

doi: 10.1080/15384101.2015.1087622

Figure Lengend Snippet: Effect of FGF/FGFR2 on the PKC-p38 pathway during expanded blastocyst formation. (A) Left, effect of the PKC inhibitor Gö6983 (1μM) on expanded blastocyst formation. Scale bar: 100 μm. Right, summary of the results. ** indicates P < 0.01 (by unpaired t-test, n = 5) compared with the control. (B) Effect of FGF2 or FGFR2 knockdown on the nuclear translocation of p-p38 MAPK and p-ERK1/2 MAPK in TEs by confocal double-immunofluorescence. Phenotype as labeled by scrambled RNA (24/30 embryos). Phenotype as labeled by FGF2 siRNA (22/29 embryos). Phenotype as labeled by FGFR2 siRNA (18/24 embryos). Scale bar: 50 μm. (C) Western blots of phosphorylated (p-) p38 (up) or ERK1/2 (down) levels in TEs in response to FGF2 or FGFR2 knockdown with siRNA transfection. (D) Effect of the p38 MAPK inhibitor SB202190 (50 µM) on the nuclear translocation of p-p38 MAPK in TEs by confocal immunofluorescence. The embryos were treated with SB202190 for 10 min. Phenotype as labeled by control (31/35 embryos). Phenotype as labeled by SB202190 treatment (27/32 embryos). Scale bar: 50 μm. (E) Western blots showing p-p38 in response to SB202190 (50 µM). (F) Effect of Gö6983 (1 μM) or SB202190 (50 µM) on the mRNA expression of protein markers of blastocysts in TEs by quantitative real-time PCR. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 (by one-way ANOVA, n = 4) compared with the corresponding controls.

Article Snippet: The embryos were washed 3 to 4 times in HTF-HEPES medium and transferred to medium containing the following: 20-μl drops of HTF + 0.2% dimethylsulfoxide (DMSO, D2650; Sigma, St. Louis, MO, USA), HTF + 1 μM of the PKC inhibitor Gö6983 (sc-203432; Santa Cruz Biotechnology, Santa Cruz, CA, USA), HTF + 50 μM of the p38 MAP kinase inhibitor SB 202190 (ab120238; Abcam, Cambridge, MA, USA), HTF + 100 ng of FGF1 antibody, HTF + 100 ng of FGF2 antibody, 100 ng of FGF4 antibody or 100 ng of FGF2 (PMG0033; Invitrogen, Gibco, Carlsbad, CA, USA) under light paraffin oil.

Techniques: Control, Knockdown, Translocation Assay, Immunofluorescence, Labeling, Western Blot, Transfection, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Pain Research

Article Title: Anxiety-induced hyperalgesia in female rats is mediated by cholecystokinin 2 receptor in rostral ventromedial medulla and spinal 5-hydroxytryptamine 2B receptor

doi: 10.2147/JPR.S187715

Figure Lengend Snippet: The experimental design and time schedule in the study. Female rats were subjected to bilateral ovariectomy and received estrogen replacement for 10 days. CCK-8/YM022 microinjection in the RVM and BW723C86/RS127445/C37H65N9O13 intrathecal injection were administrated once per day from day 6 to 8 after the estrogen replacement. The single prolonged stress (SPS) was performed on day 7. Behavioral tests were conducted on day 6 (baseline value) prior to the incision surgery (on day 7), and on days 8, 9, and 10 after the incisional surgery. Samples were collected for immunofluorescence (IF) or Western blot (WB) analysis on day 10 after behavioral tests.

Article Snippet: Experimental drugs administered intrathecally included a 5-HT2B receptor agonist (BW723C86; Tocris), a 5-HT2B receptor antagonist (RS127445; Tocris), and a PKCγ translocation inhibitor peptide (C37H65N9O13; Sigma).

Techniques: CCK-8 Assay, Injection, Immunofluorescence, Western Blot

Journal: Journal of Pain Research

Article Title: Anxiety-induced hyperalgesia in female rats is mediated by cholecystokinin 2 receptor in rostral ventromedial medulla and spinal 5-hydroxytryptamine 2B receptor

doi: 10.2147/JPR.S187715

Figure Lengend Snippet: Immunofluorescence images of 5-HT2B receptor, PKCγ and NR1 receptor in spinal dorsal horn neurons. Co-expression of 5-HT2B receptor ( A and E ), PKCγ ( B and F ), and NR1 ( C and G ) in neurons of the spinal dorsal horn in Group S+I which underwent SPS and incisional surgery. Triple immunofluorescence stained images showed that 5-HT2B/PKCγ/NR1 ( D and H ) circumscribed in the spinal cord lamina II were co-localized, as indicated by white overlay. Scale bars equal 100 μm in a-d and 20 μm in e-h.

Article Snippet: Experimental drugs administered intrathecally included a 5-HT2B receptor agonist (BW723C86; Tocris), a 5-HT2B receptor antagonist (RS127445; Tocris), and a PKCγ translocation inhibitor peptide (C37H65N9O13; Sigma).

Techniques: Immunofluorescence, Expressing, Staining

Journal: Journal of Pain Research

Article Title: Anxiety-induced hyperalgesia in female rats is mediated by cholecystokinin 2 receptor in rostral ventromedial medulla and spinal 5-hydroxytryptamine 2B receptor

doi: 10.2147/JPR.S187715

Figure Lengend Snippet: Western blot analysis of 5-HT2B receptor, PKCγ, and p-NR1 in spinal dorsal horn neurons of different groups (n=6/each). β-actin was used as an internal control. The expressions of 5-HT2B receptor, PKCγ, and p-NR1 in spinal dorsal horn in Group S+I which underwent SPS and incisional surgery were significantly higher than in other groups ( A-D ). The expression level of PKCγ in Group I was higher than in Group C. * P <0.05, ** P <0.01 compared with Group C; # P <0.05 compared with Group I; & P <0.05, && P <0.01 compared with Group S+I.

Article Snippet: Experimental drugs administered intrathecally included a 5-HT2B receptor agonist (BW723C86; Tocris), a 5-HT2B receptor antagonist (RS127445; Tocris), and a PKCγ translocation inhibitor peptide (C37H65N9O13; Sigma).

Techniques: Western Blot, Expressing

Journal: Journal of Pain Research

Article Title: Anxiety-induced hyperalgesia in female rats is mediated by cholecystokinin 2 receptor in rostral ventromedial medulla and spinal 5-hydroxytryptamine 2B receptor

doi: 10.2147/JPR.S187715

Figure Lengend Snippet: Paw withdraw mechanical threshold (PWMT) was measured at 2 hrs, 6 hrs, 24 hrs, and 48 hrs after the incisional surgery (n=6/each group). BW723C86/RS127445/C37H65N9O13 intrathecal injection was administrated and the nociceptive threshold in the Group I, Group BW723C86+I, Group RS127445+I, and Group C37H65N9O13+I decreased. In addition, the mechanical allodynia exaggerated in Group BW723C86+I which received intrathecal injections of BW723C86 and underwent incisional surgery compared with other groups ( A–D ). * P <0.05, ** P <0.01 compared with Group C; # P <0.05, ## P <0.01 compared with Group I; ▽▽ P <0.01 compared with Group BW723C86+I.

Article Snippet: Experimental drugs administered intrathecally included a 5-HT2B receptor agonist (BW723C86; Tocris), a 5-HT2B receptor antagonist (RS127445; Tocris), and a PKCγ translocation inhibitor peptide (C37H65N9O13; Sigma).

Techniques: Injection

Journal: Journal of Pain Research

Article Title: Anxiety-induced hyperalgesia in female rats is mediated by cholecystokinin 2 receptor in rostral ventromedial medulla and spinal 5-hydroxytryptamine 2B receptor

doi: 10.2147/JPR.S187715

Figure Lengend Snippet: Western blot analysis of 5-HT2B receptor, PKCγ, and p-NR1 in spinal dorsal horn neurons of different groups (n=6/each). β-actin was used as an internal control. Intrathecal injection of BW723C86 significantly upregulated the expression of 5-HT2B receptor, PKCγ, and p-NR1 in dorsal horn after incisional surgery compared with other groups ( A – D ). Group BW723C86 and Group C37H65N9O13+I showed a higher intensity of 5-HT2B receptor than the control group. The expressions level of PKCγ in Group I and Group RS127445+I was both distinctly higher than in Group C. * P <0.05, ** P <0.01 compared with Group C; # P <0.05 compared with Group I; △ P <0.05, △△ P <0.01 compared with Group BW723C86+I; ▽ P <0.05 compared with Group BW723C86.

Article Snippet: Experimental drugs administered intrathecally included a 5-HT2B receptor agonist (BW723C86; Tocris), a 5-HT2B receptor antagonist (RS127445; Tocris), and a PKCγ translocation inhibitor peptide (C37H65N9O13; Sigma).

Techniques: Western Blot, Injection, Expressing

Journal: Journal of Pain Research

Article Title: Anxiety-induced hyperalgesia in female rats is mediated by cholecystokinin 2 receptor in rostral ventromedial medulla and spinal 5-hydroxytryptamine 2B receptor

doi: 10.2147/JPR.S187715

Figure Lengend Snippet: Immunofluorescence images of 5-HT2B receptor ( A and E ), PKCγ ( B and F ) and NR1 receptor ( C and G ) in spinal dorsal horn neurons of Group RS127445+I which received intrathecal injections of RS127445 and underwent SPS and incisional surgery, and 5-HT2B receptor ( D and H ) in Group BW723C86+I which received intrathecal injections of BW723C86 and underwent incisional surgery. Scale bars equal 100 μm in a-d and 20 μm in e-h.

Article Snippet: Experimental drugs administered intrathecally included a 5-HT2B receptor agonist (BW723C86; Tocris), a 5-HT2B receptor antagonist (RS127445; Tocris), and a PKCγ translocation inhibitor peptide (C37H65N9O13; Sigma).

Techniques: Immunofluorescence

Journal: Journal of Pain Research

Article Title: Anxiety-induced hyperalgesia in female rats is mediated by cholecystokinin 2 receptor in rostral ventromedial medulla and spinal 5-hydroxytryptamine 2B receptor

doi: 10.2147/JPR.S187715

Figure Lengend Snippet: Ca 2+ channel currents have been measured in spinal cord dorsal horn neurons. The changes in I Ba ( A ), representative current traces ( B ), and the comparison of current density sets ( C ) under control conditions and during exposure to C37H65N9O13. The changes in I Ba ( D ), representative current traces ( E ), and the comparison of current density sets under control conditions, during exposure to C37H65N9O13, and during exposure to BW723C86 ( F ).

Article Snippet: Experimental drugs administered intrathecally included a 5-HT2B receptor agonist (BW723C86; Tocris), a 5-HT2B receptor antagonist (RS127445; Tocris), and a PKCγ translocation inhibitor peptide (C37H65N9O13; Sigma).

Techniques:

Journal: Frontiers in Immunology

Article Title: The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling

doi: 10.3389/fimmu.2018.00815

Figure Lengend Snippet: PKCζ is an NSM downstream effector. (A,B) Jurkat-ΔNSM and control cells were stimulated with PMA/ionomycin. Accumulation of pPKCδ, pPKCθ, and pPKCζ/λ was determined over time (A) and IL-2-release after 48 h (B) . (C) Accumulation of pPKCζ/λ was determined in Jurkat-ΔNSM and control cells stimulated with α-CD3 over time. (D) Detergent resistant membrane (DRM) domain association of PKCζ and PKCθ was determined in unstimulated (upper panels) and in 5 min α-CD3-stimulated (bottom panels) parental and Jurkat-ΔNSM cells (detection of Lck was used to identify DRM fractions). (E) PKCζ and F-actin were co-detected in CTRL (IF panels, upper row) or NSM KD cells (IF panels, bottom row) 15 min after stimulation with α-CD3-coated beads. The percentage of cells polarizing PKCζ (left graph) and relative polarization [immune synapse (IS)/cytoplasm] in individual cells were quantified (right graph). PKCζ IS localization was visualized by en face view (representative examples are shown in IF pictures on the right side). Size bars: 5 µM.

Article Snippet: When indicated Jurkat cells were pretreated with 10 μM PKCζ pseudosubstrate inhibitor (PZI) (Santa Cruz) or Jurkat-ΔNSM cells with 10 μM glycogen synthase kinase 3β (GSK3β) inhibitor indirubin-3′-monoxime (IMO) (Cayman Chemical) for 30 min before cell transfer to stimulatory slides.

Techniques: Control, Membrane

Journal: Frontiers in Immunology

Article Title: The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling

doi: 10.3389/fimmu.2018.00815

Figure Lengend Snippet: NSM is required for microtubule-organizing center (MTOC) translocation and dynamic reorganization of microtubule. (A) MTOC (detected by β-tubulin staining) immune synapse (IS) redistribution was determined in CTRL and NSM KD cells 15 min after stimulation with α-CD3-coated beads (examples shown in left panels). Quantification was based on the distance between bead center and MTOC, which should be less as 4 µm to consider MTOC as polarized (right panel). Arrows show examples of polarized MTOC in CTRL and non-polarized MTOC in NSM KD cells. Size bars: 5 µM. (B,C) Jurkat-ΔNSM and parental Jurkat cells (the latter pretreated with the PKCζ inhibitor PZI or not) were stimulated by α-CD3 on stimulatory surface coated with crosslinking antibody. MTOC structures were defined as polarized, if they were positioned in the center of cell body and recruited to the site of stimulatory surface ( xz, yz views). Summary of evaluation of at least 70 cells in z-stacks for each condition is shown in (C) . Size bars: 10 µM. (D) Accumulation of acetyl-α-tubulin was determined in CTRL and NSM KD (upper panels) or parental and Jurkat-ΔNSM cells (bottom panels) after α-CD3-stimulation over time in Western blot. (E) Detyrosinated α-tubulin was detected in α-CD3-bead stimulated CTRL and NSM KD cells (IF pictures, accumulation of detyrosinated a-tubulin at IS is depicted by arrows). Percentage of cells staining positive for MTOC with detyrosinated α-tubulin is shown in upper right graph. Only cells with detyrosinated MTOC were taken for analysis of fluorescence signal intensity by ImageJ software in lower right graph. Size bars: 5 µM.

Article Snippet: When indicated Jurkat cells were pretreated with 10 μM PKCζ pseudosubstrate inhibitor (PZI) (Santa Cruz) or Jurkat-ΔNSM cells with 10 μM glycogen synthase kinase 3β (GSK3β) inhibitor indirubin-3′-monoxime (IMO) (Cayman Chemical) for 30 min before cell transfer to stimulatory slides.

Techniques: Translocation Assay, Staining, Western Blot, Fluorescence, Software

Journal: Frontiers in Immunology

Article Title: The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling

doi: 10.3389/fimmu.2018.00815

Figure Lengend Snippet: Ceramide supplementation rescues PKCζ detergent resistant membrane (DRM) association and microtubule-organizing center (MTOC) polarization in α-CD3-stimulated NSM-depleted T cells. (A) Parental and Jurkat-ΔNSM cells were loaded with functionalized ω-C16-ceramide, DIBO488-clicked and analyzed by IF [ (A) insets] and subjected to floatation gradient centrifugation after which fractions containing DRM resident proteins Lck, LAT, CD3ζ, and CD3ε (serving as DRM markers) were identified by Western blot (upper panels) and accumulation of DIBO488 was measured in all fractions by fluorescence reader (bottom panels). (B) DRM domain association of PKCζ was determined after 5 min of α-CD3-stimulation of parental and Jurkat-ΔNSM cells pre-loaded with ω-C 16 -ceramide (detection of Lck was used to identify DRM fractions). (C) Jurkat-ΔNSM loaded with ω-C 16 -cermide or pretreated with the GSK3-inhibitor indirubin-3′-monoxime were α-CD3 stimulated. Graph shows the percentage of MTOC polarizing cells quantified after 15 min of stimulation as described in Figures B,C. Representative IF pictures for each condition are shown. Size bar: 10 µM. (A–C) One representative experiment out of three is shown.

Article Snippet: When indicated Jurkat cells were pretreated with 10 μM PKCζ pseudosubstrate inhibitor (PZI) (Santa Cruz) or Jurkat-ΔNSM cells with 10 μM glycogen synthase kinase 3β (GSK3β) inhibitor indirubin-3′-monoxime (IMO) (Cayman Chemical) for 30 min before cell transfer to stimulatory slides.

Techniques: Membrane, Gradient Centrifugation, Western Blot, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95 *

doi: 10.1074/jbc.M116.730440

Figure Lengend Snippet: PKCϵ-mediated phosphorylation of PSD-95 at serine 295 is essential for its membrane accumulation. A, immunoblot representing p-PSD-95S295 and PSD-95 expression after incubation of the different combinations of recombinant PKCϵ, PSD-95, PKCϵ activators, and PKCϵ inhibitors (inh) mentioned above at 37 °C for 10 min in vitro. B, bryostatin 1 (Bry, 0.27 nm) and DCPLA-ME (100 nm) induced the phosphorylation of PSD-95 at serine 295 position. C, expression of p-PSD-95S295 and PSD-95 and β-actin in HEK-293 cells transfected with empty vector, wild type human PSD-95, and mutant PSD-95S295K. D, expression of PSD-95, p-PSD-95S295, PKCϵ, and β-actin in the soluble (S) and particulate (P) fraction of wild-PSD-95- and PSD-95S295K-transfected HEK-293 cells treated with bryostatin 1 and DCPLA-ME for 4 h in the presence or absence of EAVSLKPT (5 μm). S, soluble fractions; P, membrane fractions. Percentage of total protein in the membrane; PKCϵ (E), PSD-95 (F) and p-PSD-95S295 (G). Data are represented as the mean ± S.E. of three independent experiments (Student's t test. *, p < 0.05; **, p < 0.005; ***, p < 0.005).

Article Snippet: Bisindolylmaleimide I (Go 6850) and PKCϵ translocation inhibitor (EAVSLKPT) were obtained from Santa Cruz Biotechnology, and SP600125 and KN-93 were obtained from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Incubation, Recombinant, In Vitro, Transfection, Plasmid Preparation, Mutagenesis

Journal: The Journal of Neuroscience

Article Title: Activin Acutely Sensitizes Dorsal Root Ganglion Neurons and Induces Hyperalgesia via PKC-Mediated Potentiation of Transient Receptor Potential Vanilloid I

doi: 10.1523/JNEUROSCI.3822-07.2007

Figure Lengend Snippet: Activin A acutely sensitizes the capsaicin-induced current in the cultured DRG neurons through the activin type I receptor ALK4 and PKC activation. A, Representative current traces from individual DRG neurons challenged by two 50 nm capsaicin (Cap) administrations separated by a 10 min treatment with vehicle, 10 ng/ml activin A, or 10 ng/ml activin plus a test reagent (additional 10 min pretreatment). The reagents tested were 20 μm SB431542 (ALK4 inhibitor), 10 μm PD98059 (MEK inhibitor), 5 μm U0126 (MEK inhibitor), 10 μm LY294002 (PI3K inhibitor), 5 μm H89 (PKA inhibitor), or 1 μm BIM (PKC inhibitor). B, Summary data (mean ± SEM) of normalized capsaicin responses (second peak current/first peak current; note change in scale after axis break) indicate that activin acts to sensitize capsaicin currents in DRG neurons through ALK4 and involves protein kinase C, but not Erk1/2, PI3K, or PKA. Additional data represent tests of activin-induced sensitization with the pseudosubstrate peptide inhibitor of PKC included in the recording pipette solution (2 μm). **p < 0.01 compared with control values. Number of observations are indicated next to each data point.

Article Snippet: The mitogen-activated protein (MAP) kinase kinase (MEK) inhibitors PD98059 and U0126, phosphoinositide 3-kinase (PI3K) inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}} LY294002 , c- Src tyrosine kinase inhibitor PP2, protein kinase A (PKA) inhibitor H89, protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM), PKC inhibitory peptide (PKC 19–31), PKCε translocation inhibitor peptide (EAVSLKPT) (PKCε TIP), negative control scrambled peptide (LSETKPAV) (PKCε NCP), and the PKCα/β inhibitor Gö6976 were all purchased from Calbiochem (San Diego, CA) and dissolved in DMSO or H 2 O as stock solutions.

Techniques: Cell Culture, Activation Assay, Transferring, Control

Journal: The Journal of Neuroscience

Article Title: Activin Acutely Sensitizes Dorsal Root Ganglion Neurons and Induces Hyperalgesia via PKC-Mediated Potentiation of Transient Receptor Potential Vanilloid I

doi: 10.1523/JNEUROSCI.3822-07.2007

Figure Lengend Snippet: Activin A causes translocation of PKCε to the cell membrane of rat DRG neurons. A, Distribution of PKCε immunoreactivity of rat DRG neurons after different treatment. Top, Confocal slice images of PKCε immunoreactivity under the indicated conditions. Bottom, Corresponding fluorescence intensity profile along a 40 μm line crossing each neuron. A uniform distribution of PKCε immunoreactivity was seen in the cytoplasm under control conditions, whereas a prominent relocation to the plasma membrane was seen for the conditions of PMA (300 nm, 2 min), BK (1 μm, 30 s), and activin (10 ng/ml, 30 s and 30 min). B, Time course of PKCε membrane translocation quantified as the percentage of neurons showing positive translocation. A positive neuron was taken as any neuron where the peak (membrane) fluorescence was ≥120% of the mean of 8 pixels in the center of the scan. At control time (no activin exposure), ∼3% of neurons showed PKCε membrane translocation, whereas at 30 s of activin exposure, ∼37% of neurons demonstrated PKCε membrane translocation, which peaked at ∼85% after 30 min. SB 431542 dramatically decreased PKCε membrane translocation. The potent PKC activator PMA (300 nm, 2 min) caused ∼91% of neurons to demonstrate PKCε membrane translocation, whereas BK (1 μm, 30 s) treatment triggered PKCε membrane translocation in ∼39% of neurons. Numbers of neurons measured are indicated for each data point.

Article Snippet: The mitogen-activated protein (MAP) kinase kinase (MEK) inhibitors PD98059 and U0126, phosphoinositide 3-kinase (PI3K) inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}} LY294002 , c- Src tyrosine kinase inhibitor PP2, protein kinase A (PKA) inhibitor H89, protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM), PKC inhibitory peptide (PKC 19–31), PKCε translocation inhibitor peptide (EAVSLKPT) (PKCε TIP), negative control scrambled peptide (LSETKPAV) (PKCε NCP), and the PKCα/β inhibitor Gö6976 were all purchased from Calbiochem (San Diego, CA) and dissolved in DMSO or H 2 O as stock solutions.

Techniques: Translocation Assay, Membrane, Fluorescence, Control, Clinical Proteomics

Journal: The Journal of Neuroscience

Article Title: Activin Acutely Sensitizes Dorsal Root Ganglion Neurons and Induces Hyperalgesia via PKC-Mediated Potentiation of Transient Receptor Potential Vanilloid I

doi: 10.1523/JNEUROSCI.3822-07.2007

Figure Lengend Snippet: Inhibition of PKCε translocation, but not PKCα activation, blocks activin A-induced TRPV1 sensitization. PKCε TIP (200 μm) added into recording pipette inhibited TRPV1 sensitization by activin, whereas PKCε NCP had no effect. Preincubation and coincubation of neurons with the PKCα/β inhibitor Gö6976 (10 nm) neither blocked activin action nor mimicked it in the absence of activin. Symbols represent mean ± SEM of the second-to-first capsaicin response ratio. **p < 0.01 relative to control; *p < 0.05 relative to activin. Numbers of observations for each condition are indicated next to each symbol.

Article Snippet: The mitogen-activated protein (MAP) kinase kinase (MEK) inhibitors PD98059 and U0126, phosphoinositide 3-kinase (PI3K) inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}} LY294002 , c- Src tyrosine kinase inhibitor PP2, protein kinase A (PKA) inhibitor H89, protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM), PKC inhibitory peptide (PKC 19–31), PKCε translocation inhibitor peptide (EAVSLKPT) (PKCε TIP), negative control scrambled peptide (LSETKPAV) (PKCε NCP), and the PKCα/β inhibitor Gö6976 were all purchased from Calbiochem (San Diego, CA) and dissolved in DMSO or H 2 O as stock solutions.

Techniques: Inhibition, Translocation Assay, Activation Assay, Transferring, Control

Journal: The American Journal of Pathology

Article Title: Co-Exposure to Cigarette Smoke and Alcohol Decreases Airway Epithelial Cell Cilia Beating in a Protein Kinase C?-Dependent Manner

doi: 10.1016/j.ajpath.2012.04.022

Figure Lengend Snippet: Effect of combination smoke and alcohol on protein kinase C (PKC)ε activity. Primary ciliated bovine bronchial epithelial cells were treated with 5% cigarette smoke extract (CSE) and 100 mmol/L ethanol (EtOH) in submerged in vitro cultures. PKCε activity was assayed at various time points from 30 minutes to 24 hours. A representative media control (M199) is indicated by the white bar. Data are shown as means ± SEs (n = 9). *P < 0.001 versus control media at matched time points of 1 to 3 hours; †P < 0.05 versus control media at matched time points of 6 to 9 hours.

Article Snippet: PKCε translocation inhibitor peptide (ε V1-2) was obtained commercially (AnaSpec, Fremont, CA).

Techniques: Activity Assay, In Vitro, Control

Journal: The American Journal of Pathology

Article Title: Co-Exposure to Cigarette Smoke and Alcohol Decreases Airway Epithelial Cell Cilia Beating in a Protein Kinase C?-Dependent Manner

doi: 10.1016/j.ajpath.2012.04.022

Figure Lengend Snippet: Effect of a protein kinase C (PKC)ε activator on cilia beat. Primary ciliated bovine bronchial epithelial cells were treated with 10 μmol/L 8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) for 30 minutes to 24 hours in submerged in vitro cultures. Ciliary beat frequency (CBF; A) was determined by Sisson-Ammons Video Analysis, and PKCε activity was determined for various concentrations (10 nmol/L to 10 μmol/L) of DCP-LA (B) and at various times (15 minutes to 24 hours) of 10 μmol/L DCP-LA (C). Media controls (M199) are indicated by white bars. Data are shown as means ± SEs (n = 9). *P < 0.01 versus control media at matched time points of 2 to 24 hours for CBF; †P < 0.05 versus control media at 30 minutes for 100 nmol/L to 10 μmol/L DCP-LA for PKCε activation; ‡P < 0.05 versus control media at matched time points of 15 to 30 minutes for 10 μmol/L DCP-LA for PKCε activation.

Article Snippet: PKCε translocation inhibitor peptide (ε V1-2) was obtained commercially (AnaSpec, Fremont, CA).

Techniques: In Vitro, Activity Assay, Control, Activation Assay

Journal: The American Journal of Pathology

Article Title: Co-Exposure to Cigarette Smoke and Alcohol Decreases Airway Epithelial Cell Cilia Beating in a Protein Kinase C?-Dependent Manner

doi: 10.1016/j.ajpath.2012.04.022

Figure Lengend Snippet: Differential effects of smoke and alcohol on basal versus ciliated cells. Both nonciliated basal (A) and ciliated (B) primary bovine bronchial epithelial cells were treated in submerged culture with M199 media (white bars), 5% cigarette smoke extract (CSE), 100 mmol/L ethanol (EtOH), individually and in combination for 1 hour, and protein kinase C (PKC)ε activity assayed. As a positive control, cells were treated with 100 ng/mL phorbol-12-myristate-13-acetate (PMA) for 15 minutes, and PKCε activity was assayed. Data are shown as means ± SEs (n = 9). *P < 0.001 versus control media for PMA treatment in both cell types and smoke+EtOH in ciliated cells.

Article Snippet: PKCε translocation inhibitor peptide (ε V1-2) was obtained commercially (AnaSpec, Fremont, CA).

Techniques: Activity Assay, Positive Control, Control

Journal: The American Journal of Pathology

Article Title: Co-Exposure to Cigarette Smoke and Alcohol Decreases Airway Epithelial Cell Cilia Beating in a Protein Kinase C?-Dependent Manner

doi: 10.1016/j.ajpath.2012.04.022

Figure Lengend Snippet: Differential effects of translocation inhibitor versus catalytic site inhibitor on ciliated cell protein kinase C (PKC)ε and motility. Ciliated primary bovine bronchial epithelial cells were treated with 10 μmol/L myristolated PKCε translocation inhibitor peptide in the presence or absence of 50 μmol/L digitonin (black bars) or 10 μmol/L active site inhibitor Ro 31-8220 and PKCε activity at 2 hours (A) or number of motile points from 1 to 6 hours (B) assayed. Data are shown as means ± SEs (n = 9). *P < 0.001 versus control media for Ro 31-8220 treatment. CBF, ciliary beat frequency.

Article Snippet: PKCε translocation inhibitor peptide (ε V1-2) was obtained commercially (AnaSpec, Fremont, CA).

Techniques: Translocation Assay, Activity Assay, Control

Journal: The American Journal of Pathology

Article Title: Co-Exposure to Cigarette Smoke and Alcohol Decreases Airway Epithelial Cell Cilia Beating in a Protein Kinase C?-Dependent Manner

doi: 10.1016/j.ajpath.2012.04.022

Figure Lengend Snippet: Localization of protein kinase C (PKC)ε directly on the isolated bovine trachea ciliary axoneme. Axonemes were visualized by differential interference contrast microscopy (A), stained with rabbit anti-PKCε antibodies (B) or nonspecific IgG (C), and visualized by confocal laser scanning microscopy. Axonemes were also assayed for PKCε activity in the presence or absence of calcium, lipid, substrate, or dithiothrietol (DTT) (D).

Article Snippet: PKCε translocation inhibitor peptide (ε V1-2) was obtained commercially (AnaSpec, Fremont, CA).

Techniques: Isolation, Microscopy, Staining, Confocal Laser Scanning Microscopy, Activity Assay

Journal: The American Journal of Pathology

Article Title: Co-Exposure to Cigarette Smoke and Alcohol Decreases Airway Epithelial Cell Cilia Beating in a Protein Kinase C?-Dependent Manner

doi: 10.1016/j.ajpath.2012.04.022

Figure Lengend Snippet: Effect of combination smoke and alcohol on cilia in lung slices. Precision-cut mouse lung slices were treated with 5% cigarette smoke extract (CSE) and 100 mmol/L ethanol (EtOH) in submerged in vitro culture. Ciliary beat frequency (CBF; A) and the average number of motile points per field of cells (B) were determined by Sisson-Ammons Video Analysis from 1 to 21 hours. Protein kinase C (PKC)ε activity was assayed from 1 to 6 hours (C). Representative media controls (M199) are indicated by the white bars. Data are shown as means ± SEs (n = 9). *P < 0.05 versus control media at matched time points of 3 to 21 hours for CBF; †P < 0.01 versus control media at matched time points of 4 to 21 hours for average motile points; ‡P < 0.001 versus control media at matched time points of 1 to 3 hours.

Article Snippet: PKCε translocation inhibitor peptide (ε V1-2) was obtained commercially (AnaSpec, Fremont, CA).

Techniques: In Vitro, Activity Assay, Control

Journal: The American Journal of Pathology

Article Title: Co-Exposure to Cigarette Smoke and Alcohol Decreases Airway Epithelial Cell Cilia Beating in a Protein Kinase C?-Dependent Manner

doi: 10.1016/j.ajpath.2012.04.022

Figure Lengend Snippet: Effect of in vivo smoke and alcohol exposure on cilia. Mice were either sham-treated (white bars) with air and water, whole body exposed to cigarette smoke (smoke), fed 20% alcohol (EtOH), or exposed to both alcohol and cigarette smoke in combination (smoke+EtOH) for 8 weeks. Ciliary beat frequency (CBF; A) and protein kinase C (PKC)ε activity (B) were assayed from tracheal epithelium. Data are shown as means ± SEs (n = six mice per group). *P < 0.01 for smoke+EtOH-treated versus sham-treated mice.

Article Snippet: PKCε translocation inhibitor peptide (ε V1-2) was obtained commercially (AnaSpec, Fremont, CA).

Techniques: In Vivo, Activity Assay

Journal: The American Journal of Pathology

Article Title: Co-Exposure to Cigarette Smoke and Alcohol Decreases Airway Epithelial Cell Cilia Beating in a Protein Kinase C?-Dependent Manner

doi: 10.1016/j.ajpath.2012.04.022

Figure Lengend Snippet: Sequence of in vivo smoke and alcohol co-exposure does not alter the effects on protein kinase C (PKC)ε. Mice were either whole-body smoke-exposed first followed by alcohol feeding (gray bars) or alcohol-fed first followed by whole-body smoke exposure (black bars) in vivo, and PKCε activity was measured in both tracheal epithelium (A) and precision-cut lung slices (B). Data are shown as means ± SEs (n = 6 mice/group). *P < 0.01 for either smoke then EtOH-treated or EtOH then smoke-treated versus sham-treated mice.

Article Snippet: PKCε translocation inhibitor peptide (ε V1-2) was obtained commercially (AnaSpec, Fremont, CA).

Techniques: Sequencing, In Vivo, Activity Assay

Journal: The American Journal of Pathology

Article Title: Co-Exposure to Cigarette Smoke and Alcohol Decreases Airway Epithelial Cell Cilia Beating in a Protein Kinase C?-Dependent Manner

doi: 10.1016/j.ajpath.2012.04.022

Figure Lengend Snippet: Effect of smoke and alcohol exposure on cilia from protein kinase C (PKC)ε knockout mice. Tracheal rings and lung slices were cut from mice that lacked PKCε expression. Change in ciliary beat frequency (CBF) in response to ex vivo 10 μmol/L Ro 31-8220 treatment (versus baseline CBF) in the trachea of wild-type, PKCε knockout (PKCεKO), and PKCδ knockout (PKCδ-KO) mice were assayed by Sisson-Ammons Video Analysis (A). Lung slice PKCε activity in response to ex vivo treatment with 10 μmol/L 8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) was assayed in wild-type (WT) and PKCεKO mice (B). Lung slice PKCε activity in response to in situ treatment with smoke and alcohol was assayed in WT and PKCεKO mice (C). Data are shown as means ± SEs (n = 6). *P < 0.01 for changes in CBF in WT and PKCδKO mice in the presence versus absence of Ro 31-8220; †P < 0.001 for PKCε activation in WT versus PKCεKO mice in response to DCP-LA; ‡P < 0.001 for PKCε activation in WT versus PKCεKO mice in response to the combination of smoke+alcohol. EtOH, ethanol.

Article Snippet: PKCε translocation inhibitor peptide (ε V1-2) was obtained commercially (AnaSpec, Fremont, CA).

Techniques: Knock-Out, Expressing, Ex Vivo, Activity Assay, In Situ, Activation Assay