Journal:

Article Title: Role of p38 Mitogen-Activated Protein Kinase in Middle Ear Mucosa Hyperplasia during Bacterial Otitis Media

doi: 10.1128/IAI.72.8.4662-4667.2004

Figure Lengend Snippet: Western blot of total (A) and phosphorylated (B) p38 in middle ear mucosa at various postinoculation times. Total protein was equalized in all lanes. Quantitative analysis of total p38 (C) shows a gradual increase over time, while the ratio of phospho-p38 to total p38 (D) shows strong p38 phosphorylation 1 to 6 h after bacterial inoculation, with lower levels thereafter. Higher levels of intensity observed for phospho-p38 than for total p38 at some time points presumably reflect differential sensitivity of the antibodies used, blotting efficiency, and the fact that total p38 was measured on stripped blots.

Article Snippet: After being transferred to polyvinylidene difluoride paper, the proteins were immunoblotted with rabbit polyclonal antibody that reacted only with phosphorylated p38 kinase (Promega, Madison, Wis.).

Techniques: Western Blot

Journal:

Article Title: Role of p38 Mitogen-Activated Protein Kinase in Middle Ear Mucosa Hyperplasia during Bacterial Otitis Media

doi: 10.1128/IAI.72.8.4662-4667.2004

Figure Lengend Snippet: Western blot of total and phosphorylated p38 in middle ear mucosal explants harvested 48 h after injection of saline or bacterial inoculation and cultured for 72 hours. Total protein was equalized in all lanes.

Article Snippet: After being transferred to polyvinylidene difluoride paper, the proteins were immunoblotted with rabbit polyclonal antibody that reacted only with phosphorylated p38 kinase (Promega, Madison, Wis.).

Techniques: Western Blot, Injection, Cell Culture

Journal: The Journal of Neuroscience

Article Title: p38 MAP Kinase Mediates Both Short-Term and Long-Term Synaptic Depression in Aplysia

doi: 10.1523/JNEUROSCI.23-19-07317.2003

Figure Lengend Snippet: Characterization of Aplysia p38 MAP kinase. A, A phylogenetic comparison using clustal analysis with the DNA-Star program shows that the Aplysia enzyme belongs to the α-β subfamily of p38 kinases. B, The activated mammalian p38 kinase upstream kinase, MKK6, can phosphorylate recombinant Ap-p38 kinase in vitro. The phosphorylated enzyme was detected with a commercial anti-human phospho-p38 (Pp38) antibody, and the total amount of kinase was detected with an anti-Ap-p38 antibody (Fig. 2). C, When phosphorylated by MKK6, the recombinant Ap-p38 kinase can phosphorylate human ATF2 in vitro, which was detected with an anti-phospho ATF2 antibody, and the phosphorylation of ATF2 was blocked by the p38 kinase inhibitor SB 203580 (0.5 μm). Inactivated Ap-p38 kinase did not phosphorylate ATF2. D, Inhibition by SB 203580 was dose dependent. The same amount of ATF2 was incubated with activated Ap-p38 kinase and varying concentrations of the inhibitor. The phosphorylation of ATF2 was detected by anti-phospho-ATF2 antibody, and the activity of p38 kinase was assessed densitometrically by the phosphorylation of ATF2. With increasing concentrations of SB 203580, kinase activity was gradually inhibited with an IC50 of ∼50 nm.

Article Snippet: Human ATF2 (2 μg) (Cell Signaling Technology) was incubated with phosphorylated Ap-p38 kinase in kinase buffer (Cell Signaling Technology) supplemented with 0.2 m m ATP at 30°C for 30 min. Proteins from the reaction mixture were separated by SDS-PAGE, and the phosphorylated transcription factor was quantified by immunoblotting using anti-phospho-ATF2 (Cell Signaling Technology).

Techniques: Recombinant, In Vitro, Inhibition, Incubation, Activity Assay

Journal: The Journal of Neuroscience

Article Title: p38 MAP Kinase Mediates Both Short-Term and Long-Term Synaptic Depression in Aplysia

doi: 10.1523/JNEUROSCI.23-19-07317.2003

Figure Lengend Snippet: Antibody against Aplysia p38 kinase. A, An affinity-purified antibody raised against a peptide (PEAEKYDQSFEEMELG) from the Aplysia kinase specifically recognized a single component of approximately Mr 38 kDa, the size expected of p38 kinase, from homogenates of Aplysia pleural ganglia. Preincubation of the antibody with the peptide used as immunogen blocked the signal. B, Recombinant Ap-p38 kinase activated by mammalian MKK6 was incubated with human ATF2 and ATP, with or without Ap-p38 antibody. Kinase activity, monitored by the phosphorylation of ATF2 by Western blot with anti-phospho-ATF2 antibody, was completely blocked by the Ap-p38 antibody.

Article Snippet: Human ATF2 (2 μg) (Cell Signaling Technology) was incubated with phosphorylated Ap-p38 kinase in kinase buffer (Cell Signaling Technology) supplemented with 0.2 m m ATP at 30°C for 30 min. Proteins from the reaction mixture were separated by SDS-PAGE, and the phosphorylated transcription factor was quantified by immunoblotting using anti-phospho-ATF2 (Cell Signaling Technology).

Techniques: Affinity Purification, Recombinant, Incubation, Activity Assay, Western Blot

Journal: The Journal of Neuroscience

Article Title: p38 MAP Kinase Mediates Both Short-Term and Long-Term Synaptic Depression in Aplysia

doi: 10.1523/JNEUROSCI.23-19-07317.2003

Figure Lengend Snippet: p38 kinase is bidirectionally regulated by 5-HT and FMRFa. Treatment with FMRFa (9 μm, 10 min) increased the phosphorylation of p38 kinase in pleural ganglion neurons, and treatment with 5-HT (50 μm, 10 min) decreased the phosphorylation. As control, the total amount of kinase was detected by immunoblotting with anti-Ap-p38 kinase. A, Representative immunoblots from four independent experiments are shown. B, Group data showing the regulation of phosphorylation by the two transmitters. Treatment with FMRFa stimulated the phosphorylation of p38 kinase, and 5-HT diminished the phosphorylation. The ratio of phospho-p38 kinase to the nonphosphorylated form was determined in each group of four experiments. The bars represent the mean values obtained with each transmitter divided by the contral ratio. Asterisks indicate significance p < 0.05.

Article Snippet: Human ATF2 (2 μg) (Cell Signaling Technology) was incubated with phosphorylated Ap-p38 kinase in kinase buffer (Cell Signaling Technology) supplemented with 0.2 m m ATP at 30°C for 30 min. Proteins from the reaction mixture were separated by SDS-PAGE, and the phosphorylated transcription factor was quantified by immunoblotting using anti-phospho-ATF2 (Cell Signaling Technology).

Techniques: Western Blot

Journal: The Journal of Neuroscience

Article Title: p38 MAP Kinase Mediates Both Short-Term and Long-Term Synaptic Depression in Aplysia

doi: 10.1523/JNEUROSCI.23-19-07317.2003

Figure Lengend Snippet: Ap-p38 kinase mediates short-term depression by releasing arachidonate. A, Ap-p38 kinase mediates short-term depression. The cells were treated with one 5 min pulse of FMRFa (1×F), which depressed the EPSP recorded at 5 min (-50.9 ± 10.6%; n = 8). Preincubation with the p38 kinase inhibitor SB 203580 (3 μm, 30 min) blocked the FMRFa-induced depression (1×F + SB; -12.7 ± 7.5%; n = 15; p < 0.05). The inhibitor alone (SB alone) had no short-term effect on the EPSP (-7.3 ± 5.6%; n = 7). Data were analyzed by ANOVA. Histograms show the percentage changes in the mean (±SE) of the EPSP amplitudes 5 min after the treatment. B, Ap-p38 kinase mediates the release of arachidonate. The FMRFa-induced release of arachidonate in pleural ganglia (Control, 1944.6 ± 305.7 pg, n = 7; FMRFa, 3658 ± 696.4 pg, n = 7) was blocked by 1 μm SB 203580 (FMRFa + SB, 1421 ± 249.3 pg, n = 6, p < 0.01). Data were analyzed by ANOVA.

Article Snippet: Human ATF2 (2 μg) (Cell Signaling Technology) was incubated with phosphorylated Ap-p38 kinase in kinase buffer (Cell Signaling Technology) supplemented with 0.2 m m ATP at 30°C for 30 min. Proteins from the reaction mixture were separated by SDS-PAGE, and the phosphorylated transcription factor was quantified by immunoblotting using anti-phospho-ATF2 (Cell Signaling Technology).

Techniques:

Journal: The Journal of Neuroscience

Article Title: p38 MAP Kinase Mediates Both Short-Term and Long-Term Synaptic Depression in Aplysia

doi: 10.1523/JNEUROSCI.23-19-07317.2003

Figure Lengend Snippet: Bidirectional function of p38 kinase. A, SB 203580 blocked long-term depression and enhanced long-term facilitation: representative examples of EPSP traces before and 24 hr after treatment, and summary data. To induce long-term depression, the sensory-to-motor neuron cocultures were treated with five 5 min pulses of 1 μm FMRFa (5× F), which resulted in depressed EPSPs at 24 hr (-26.8 ± 6.6%; n = 12). This long-term depression was blocked by incubation with SB 203580 (3 μm) 30 min before and throughout the treatment with FMRFa (5×F+ SB; 12.6 ± 10.8%; n = 10; p < 0.05). For long-term facilitation, the cells were treated with five 5 min pulses or a single 5 min pulse of 5-HT (10 μm). A single pulse of 5-HT (1× S) had no long-term effect (10.6 ± 15.3%; n = 7), but long-term facilitation was established in cells preincubated with the inhibitor (3 μm, 30 min) and then treated with one pulse of 5-HT (1× S + SB; 88.9 ± 22.5; n = 10; p < 0.01). The long-term facilitation produced in this way was comparable with that resulting from five pulses of 5-HT (5× S; 103.5 ± 7.5%; n = 8). Five pulses of 5-HT in cells incubated in 3 μm SB 203580 (5× S + SB) did not enhance the long-term facilitation further (102.2 ± 18.7%; n = 7). Cells treated with the inhibitor alone (SB alone; 3.1 ± 4.7%; n = 11) had no long-term effect. B, Injection of Ap-p38 antibody blocked long-term depression and enhanced long-term facilitation: EPSP traces and summary data. The inactivating antibody against Ap-p38 kinase (Fig. 2) was injected into pleural sensory neurons before treatment with FMRFa or 5-HT. Five 5 min pulses of FMRFa coupled with injection of the vehicle (0.4 m potassium acetate and 10 mm Tris-HCl, pH 7.4) (5× F + vehicle) induced long-term depression at 24 hr (-24.2 ± 5.4%; n = 13); the long-term depression was blocked by injecting the antibody (5× F +αAp-p38; 3.3 ± 10.9%; n = 9; p < 0.05). A single pulse of 5-HT coupled with injection of the vehicle (1× S + vehicle) had no long-term effect (8.9 ± 3.7%; n = 14) but produced long-term facilitation after the Ap-p38 antibody was injected (1× S +αAp-p38; 81.2 ± 22.2%; n = 7; p < 0.01). No effect on the EPSP at 24 hr was observed with the injection of vehicle alone (12.5 ± 11.2%; n = 4) or antibody alone (4.1 ± 13.3%; n = 7). C, Injection of activated Ap-p38 kinase enhanced long-term depression and blocked long-term facilitation: EPSP traces and summary data. A single pulse of FMRFa coupled with the injection of vehicle (1× F + vehicle) had no long-term effect (7.6 ± 5.2%; n = 8). Injection of phospho-Ap-p38 kinase into sensory neurons coupled with a single pulse of FMRFa (1×F + p-Ap-p38) induced long-term depression (-24.6 ± 5.91%; n = 11; p < 0.01). Five pulses of 5-HT coupled with injection of vehicle (5× S + vehicle) induced long-term facilitation (127.5 ± 29.7%; n = 13); long-term facilitation was blocked by injecting phospho-Ap-p38 kinase (5× S + p-Ap-p38; 38.3 ± 12.5; n = 13%; p < 0.01). No effect at 24 hr was observed with injection of vehicle alone (-12.3 ± 9.2%; n = 6) or injection of phospho-Ap-p38 kinase alone (3.3 ± 6.8%; n = 12). Data were analyzed by ANOVA. Histograms show percentage changes in the mean (±SE) of EPSP amplitudes 24 hr after the treatment.

Article Snippet: Human ATF2 (2 μg) (Cell Signaling Technology) was incubated with phosphorylated Ap-p38 kinase in kinase buffer (Cell Signaling Technology) supplemented with 0.2 m m ATP at 30°C for 30 min. Proteins from the reaction mixture were separated by SDS-PAGE, and the phosphorylated transcription factor was quantified by immunoblotting using anti-phospho-ATF2 (Cell Signaling Technology).

Techniques: Incubation, Produced, Injection

Journal: The Journal of Neuroscience

Article Title: p38 MAP Kinase Mediates Both Short-Term and Long-Term Synaptic Depression in Aplysia

doi: 10.1523/JNEUROSCI.23-19-07317.2003

Figure Lengend Snippet: Ap-p38 kinase activates CREB2 and induces histone deacetylation. A, Ap-p38 kinase phosphorylates CREB2. GST or the GST-CREB2 fusion protein was incubated with activated Ap-p38 and γ-labeled 32P-ATP at 30°C for 30 min. The products were analyzed by running a gel and then exposing to an x-ray film. The kinase phosphorylated the GST-CREB2 fusion protein but not GST. B, Ap-p38 kinase activity is critical for recruiting CREB2 and HDAC5 to the C/EBP promoter. With ChIP assays, a basal level of CREB2 and HDAC5 was detected at the promoter in unstimulated neurons. Treatment with FMRFa (10 μm) for 90 min induced more CREB2 and HDAC5 to bind. SB 203580 (1 μm) lowered the basal amount of both CREB2 and HDAC5, an effect similar to that of a 90 min treatment with 5-HT. The inhibitor also blocked the FMRFa-induced recruitment of CREB2 and HDAC5. As control, chromatin samples were also analyzed before immunoprecipitation (Input) to show that equal amounts of starting material were applied. C, Ap-p38 kinase activity is necessary for FMRFa to induce histone deacetylation. Basal histone H4 acetylation (Acetyl H4) was detected at the C/EBP promoter. Although treatment with 5-HT (S) increased H4 acetylation, FMRFa, either alone (F) or together with 5-HT (FS), decreased the acetylation. SB 203580 blocked the FMRFa-induced histone deacetylation at the promoter (Acetyl H4/SB 203580). In addition, 5-HT (S) induced the specific acetylation of lysine 8 of H4 [Acetyl H4 (K8)], and FMRFa cotreatment (FS) blocked this acetylation. SB 203580 blocked this inhibitory effect of FMRFa [Acetyl H4 (K8)/SB 203580]. As control, chromatin samples were also analyzed before immunoprecipitation (Input). All of the samples were also analyzed with the primers specific to the promoter of Aplysia histone H4, a gene with a strong basal expression but no response to either 5-HT or FMRF (Guan et al., 2002). No changes were observed after any of the treatments (data not shown).

Article Snippet: Human ATF2 (2 μg) (Cell Signaling Technology) was incubated with phosphorylated Ap-p38 kinase in kinase buffer (Cell Signaling Technology) supplemented with 0.2 m m ATP at 30°C for 30 min. Proteins from the reaction mixture were separated by SDS-PAGE, and the phosphorylated transcription factor was quantified by immunoblotting using anti-phospho-ATF2 (Cell Signaling Technology).

Techniques: Incubation, Labeling, Activity Assay, Immunoprecipitation, Expressing

Journal: The Journal of Neuroscience

Article Title: p38 MAP Kinase Mediates Both Short-Term and Long-Term Synaptic Depression in Aplysia

doi: 10.1523/JNEUROSCI.23-19-07317.2003

Figure Lengend Snippet: p38 MAP kinase regulates synaptic plasticity bidirectionally. A, In the cytoplasm, the inhibitory neuropeptide FMRFa, through an as yet unidentified mechanism, activates p38 kinase. The facilitatory neurotransmitter 5-HT, on the other hand, inhibits the kinase, presumably through activation of PKA. B, Activated p38 kinase moves into the nucleus, where it phosphorylates transcription factors CREB2 and ATF2. Phosphorylated CREB2 binds to the C/EBP promoter and induces histone deacetylation to repress the expression of C/EBP. Thus the long-term facilitation induced by 5-HT is blocked. Phosphorylated CREB2 and ATF2, which are also transcription activators, can bind to promoters of yet unidentified genes to induce the expression of proteins important to long-term depression, presumably through induction of histone acetylation. As a result, long-term depression develops.

Article Snippet: Human ATF2 (2 μg) (Cell Signaling Technology) was incubated with phosphorylated Ap-p38 kinase in kinase buffer (Cell Signaling Technology) supplemented with 0.2 m m ATP at 30°C for 30 min. Proteins from the reaction mixture were separated by SDS-PAGE, and the phosphorylated transcription factor was quantified by immunoblotting using anti-phospho-ATF2 (Cell Signaling Technology).

Techniques: Activation Assay, Expressing

Journal:

Article Title: Signaling Cascades Triggered by Bacterial Metabolic End Products during Reactivation of Kaposi's Sarcoma-Associated Herpesvirus

doi: 10.1128/JVI.02504-06

Figure Lengend Snippet: (A) Effect of p38 and PI3K inhibition on the induction of early lytic KSHV gene expression. BCBL-1 cells were pretreated with the PI3 kinase (LY294002) or p38 (SB202190 and PD169316) inhibitors for 1 h at 37°C and then treated with spent medium from S. aureus or P. gingivalis for 24 or 48 h. The expression of early lytic (vIL-6), phospho-p38 (p∼p38), total p38, and cellular β-actin proteins was examined by immunoblot analysis. PD, PD169316; SB, SB202190; LY, LY294002; PG, P. gingivalis; SA, S. aureus. (B) Effect of p38 inhibition on the induction of late lytic KSHV gene expression. BCBL-1 cells were pretreated with the p38 inhibitor (SB202190) for 1 h at 37°C and induced with spent media from P. gingivalis (PG) and F. nucleatum (FN). The expression of late lytic (K8.1) and cellular β-actin proteins was examined by immunoblot analysis using anti-K8.1 and anti-β-actin antibodies.

Article Snippet: The rabbit polyclonal antibody detecting phosphorylated kinase p38 and the respective inactive nonphosphorylated form were purchased from Cell Signaling Technology, Beverly, MA.

Techniques: Inhibition, Expressing, Western Blot

Journal:

Article Title: Signaling Cascades Triggered by Bacterial Metabolic End Products during Reactivation of Kaposi's Sarcoma-Associated Herpesvirus

doi: 10.1128/JVI.02504-06

Figure Lengend Snippet: Effect of p38 inhibition on the hyperacetylation of H3 and induction of late KSHV gene expression. Whole-cell protein was extracted from BCBL-1 cells pretreated with the p38 inhibitor (SB202190) for 1 h at 37°C and induced with spent media from P. gingivalis (PG) and F. nucleatum (FN). Extracts were run on an 18% SDS-polyacrylamide gel. The expression of early lytic vIL-6, the acetylation status of H3, and the cellular β-actin protein were examined by immunoblot analysis using anti-vIL-6, anti-acetyl H3, and anti-β-actin antibodies, respectively. SB, SB202190.

Article Snippet: The rabbit polyclonal antibody detecting phosphorylated kinase p38 and the respective inactive nonphosphorylated form were purchased from Cell Signaling Technology, Beverly, MA.

Techniques: Inhibition, Expressing, Western Blot

Journal:

Article Title: Signaling Cascades Triggered by Bacterial Metabolic End Products during Reactivation of Kaposi's Sarcoma-Associated Herpesvirus

doi: 10.1128/JVI.02504-06

Figure Lengend Snippet: Model for induction of KSHV reactivation by metabolic end products from gram-negative bacteria, which contain high levels of butyric acid, inhibit cellular HDACs, and activate the p38 kinase pathway. Activation of the p38 pathway may lead to phosphorylation of cellular histones, which results in increased sensitivity to acetylation by histone acetyltransferases. The combined effects of HDAC inhibition and increased acetylation sensitivity result in hyperacetylation of the histones on immediate early viral promoters. The acetylation event neutralizes the positive charge of the histone tail and remodels chromatin structure, making the nucleosome accessible for binding to transcription factors and activation of the temporal cascade of viral gene expression and subsequent viral production. Viral genes are denoted as E (early), IE (immediate early), and L (late). MEK, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase; SB, SB202190; PD, PD169316.

Article Snippet: The rabbit polyclonal antibody detecting phosphorylated kinase p38 and the respective inactive nonphosphorylated form were purchased from Cell Signaling Technology, Beverly, MA.

Techniques: Activation Assay, Inhibition, Binding Assay, Expressing