Display Settings:

Items per page

Results: 6

1.
FIGURE 6

FIGURE 6. From: MAPK p38 Regulates Transcriptional Activity of NF-?B in Primary Human Astrocytes via Acetylation of p65.

Signals arising from cytokine receptors (hypothetically shown together), trigger the canonical pathway of NF-κB activation as is shown on the right side. This is facilitated by one or more kinase(s) that phosphorylate p65. Additional signals activate MAPK p38 (left) which then phosphorylates and activates coactivator p300. Thus activated, p300 binds to NF-κB and acetylates the critical K310 residue of p65. As a result, optimal transcriptional ability is achieved by the NF-κB-p300 transcriptional complex.

Ramendra N. Saha, et al. J Immunol. ;179(10):7101-7109.
2.
FIGURE 5

FIGURE 5. MAPK p38 regulates acetylation of p65-K310 in human primary astrocytes. From: MAPK p38 Regulates Transcriptional Activity of NF-?B in Primary Human Astrocytes via Acetylation of p65.

A, Cells plated in a 100-mm dish were transfected with 4 µg of an empty vector or Δp300. Twenty-four hours after transfection, cells were stimulated with IL-IF under serum-free condition. After 1 h of stimulation, nuclear lysates were immunoprecipitated with anti-p65 Ab and immunoprecipitates were probed with Abs against Ac-K310-p65. The same membrane was stripped and immunoblotted against p65. B, Cells pretreated with SB for 1 h were stimulated with IL-IF for another hour under serum-free conditions. Then nuclear lysates were immunoprecipitated anti-p65 Ab and immunoprecipitates were probed with Abs against Ac-K310-p65 and p65. C, Immuno-cytochemical staining of Ac-K310-p65 in cells treated as in B. DAPI staining is included to visualize the nuclear localization of anti-Ac-K310-p65 signal. D, Cells plated in a 100-mm dish were transfected with 4 µg of an empty vector or kinase deficient Δp38. Twenty-four h after transfection, cells were stimulated with IL-IF under serum-free condition. After 1 h of stimulation, nuclear lysates were immunoprecipitated with anti-p65 Ab and immunoprecipitates were probed with Abs against Ac-K310-p65 and p65. Results represent three independent experiments.

Ramendra N. Saha, et al. J Immunol. ;179(10):7101-7109.
3.
FIGURE 4

FIGURE 4. MAPK p38 phosphorylates p300 and regulates its acetyltransferase activity in human primary astrocytes. From: MAPK p38 Regulates Transcriptional Activity of NF-?B in Primary Human Astrocytes via Acetylation of p65.

A, Metabolic immuno-phospho-labeling assay was performed as described under Materials and Methods to detect the phosphorylation of p300. [32P]Orthophosphate-labeled cells were treated with SB for 1 h, followed by stimulation with IL-IF. Then nuclear lysates were immunoprecipitated with Abs against p300 and immunoprecipitates were electrophoresed. The gel was dried and exposed to autoradiography. B, Cells plated in a 100-mm dish were transfected with 4 µg of an empty vector or kinase deficient Δp38. Twenty-four hours after transfection, cells were labeled and stimulated with IL-IF as described above. Then p300 immunoprecipitates from nuclear lysates were electrophoresed and autoradiographed. In both cases (A and B), to verify equivalent loading, the gel was rehydrated and subsequently immunoblotted with anti-p300. C, Cells pretreated with SB for 1 h were stimulated with IL-IF for another hour under serum-free condition. Then, nuclear lysates were immunoprecipitated with Abs against p300 and activity of HAT was assayed in immunoprecipitates as described above. Data are mean ± SD of three different experiments.*, p< 0.05 vs control.

Ramendra N. Saha, et al. J Immunol. ;179(10):7101-7109.
4.
FIGURE 2

FIGURE 2. NF-κB p65 phosphorylation status is not sensitive to p38 inhibitor in human primary astrocytes. From: MAPK p38 Regulates Transcriptional Activity of NF-?B in Primary Human Astrocytes via Acetylation of p65.

A, Astrocytes were serum starved and subsequently pretreated with 10 µM of SB followed by IL-IF stimulation for 1 h. Nuclear lysate was obtained from these astrocytes and was used to perform coimmunoprecipitation assay with anti-p65 Ab. Immunoprecipitate thus obtained was immunoblotted with Abs against phosphoserine. The same blot was stripped and probed for p65. IgG H chain (IgG-H) is shown as loading control. B, Coimmunoprecipitation was performed as in (A) and the immunoprecipitate was probed with anti-phospho-S276-p65 Ab. After stripping, the same blot was also probed with anti-p65 Abs. C, Cells treated as in A were used for immunofluorescence with anti-phospho p65-S276 Ab. Signals (red) were detected with Cy5 tagged secondary Ab. Bar = 10 µm. DAPI was used to visualize the nucleus. D, Astrocytes were serum starved and subsequently pretreated with 10 µM each of H89 and U0126 for an hour before treatment with IL-IF for an additional hour. Coimmunoprecipitation was performed as in A and the immunoprecipitate was probed with anti-phospho-S276-p65 Ab. After stripping, the same blot was also probed with anti-p65 Abs. E, Cells were transfected either empty vector or with Δp38. Next day, these cells were serum starved and were then treated with IL-IF for 1 h. Coimmunoprecipitation was performed as in A and subsequently blotting was performed as in D. F, Cells were transfected either with 100 nM si-C or si-p38α. After 48 h, they were serum starved and treated with IL-IF for an hour and coimmunoprecipitation was performed as in A and the immunoprecipitate was probed with anti-phospho-S536-p65 Ab. After stripping, the same blot was also probed with anti-p65 Abs. G, Cells were transfected with FLAG-p65 (Δ313). Twenty-four hours after transfection, cells were treated with SB for 1 h followed by stimulation with IL-IF under serum-free condition. After 1 h, cells were fixed and used for immunofluorescence studies with anti-FLAG (red) and anti-phospho-S276-p65 (green) Abs. Bar = 20 µm. DAPI was used to visualize nucleus. Results represent three independent trials.

Ramendra N. Saha, et al. J Immunol. ;179(10):7101-7109.
5.
FIGURE 3

FIGURE 3. Interaction of NF-κB p65 with coactivator p300 is sensitive to p38 inhibitor in human primary astrocytes. From: MAPK p38 Regulates Transcriptional Activity of NF-?B in Primary Human Astrocytes via Acetylation of p65.

A, Cells were treated as in Fig. 2A. Nuclear lysates obtained from these cells were immunoprecipitated with anti-p65 Ab and HAT assay were performed in immunoprecipitates as described under Materials and Methods. Data are mean ± SD of three different experiments. **, p < 0.01 vs control. B, Cells were cotransfected with 0.1 µg of either empty vector, p300 expression plasmid, or dominant-negative p300 (Δp300) and 0.1 µg of phiNOS(7.2)Luc and 10 ng of pRL-TK. After 24 h of transfection, cells were stimulated with IL-IF for 12 h. Firefly and Renilla luciferase activities were analyzed in total cell extract. Data are mean ± SD of three different experiments.*, p < 0.05 vs IL-IF + vector. C, Cells plated in 100-mm dishes were cotransfected with T7-p65 (3 µg) and FLAG-p300 (3 µg). After 24 h of transfection, cells were treated with different doses of SB for 1 h followed by stimulation with IL-IF for another hour. Nuclear extracts were immunoprecipitated with Abs against FLAG and the resulting immunoprecipitate was immunoblotted with Abs against T7. The same blot was stripped and probed for FLAG. Lower panels, Whole cell extract from a quarter of similarly transfected cells used for IPs above was Western blotted to detect pre-IP level of indicated ectopic proteins. D, Cells pretreated with 10 µM SB for 1 h were stimulated with IL-IF for another hour under serum-free condition. Nuclear extracts isolated from these cells were immunoprecipitated with Abs against either p65 (upper panels) or p300 (lower panels). The p65 immunoprecipitate was gel separated and was probed for p300 (upper panels). The same blot was probed for p65 after stripping. In contrast, the p300 immunoprecipitate was probed for p65 (lower panels). The same blot was also stripped and probed for p300. E, Cells were transfected either with 100 nM si-C or si-p38α. After 48 h, cells were serum starved and treated with IL-IF for an hour. Nuclear lysate was obtained from these astrocytes and was used to perform coimmunoprecipitation assay with anti-p65 Ab. Immunoprecipitate thus obtained was immunoblotted with anti-p300. The same blot was stripped and probed for p65. F, Cells were treated as in D. Nuclear extracts isolated from these cells were immunoprecipitated with anti-p300. Immunoprecipitate thus obtained was immunoblotted with anti-phospho-S276-p65. The same blot was stripped and was reprobed for p38α and p300. Results represent three independent experiments.

Ramendra N. Saha, et al. J Immunol. ;179(10):7101-7109.
6.
FIGURE 1

FIGURE 1. NF-κB transcriptional activity, but not DNA binding activity, is sensitive to p38 inhibitor in human primary astrocytes. From: MAPK p38 Regulates Transcriptional Activity of NF-?B in Primary Human Astrocytes via Acetylation of p65.

A, Serum-starved cells (6 h) were pretreated with indicated doses of SB for 1 h and then stimulated with TNF-α (10 ng/ml), IL-1β (10 ng/ml), or the combination of IL-1β (10 ng/ml) and IFN-γ (25 U/ml) (IL-IF) for another hour. Nuclear extracts prepared from these cells were used to perform EMSA (Promega Probe) and supershift assay. B, Nuclear lysate obtained from serum-starved cells pretreated with 10 µM of SB followed by IL-IF treatment for various time periods was immunoblotted with Abs against p65 and LaminB. C, Cells were cotransfected with 10 ng of pRL-TK and 0.2 µg of pBIIX-Luc using Lipofectamine Plus as described under Materials and Methods. After 24 h of transfection, cells were incubated with different concentrations of SB for 1 h followed by stimulation with IL-IF for 6 h. Firefly and Renilla luciferase activities were analyzed in total cell extract. D, Cells were cotransfected with 0.2 µg of phiNOS(7.2)Luc (an 7.2-kb human iNOS promoter-driven reporter construct) and 10 ng of pRL-TK (transfection efficiency control). After 24 h of transfection, cells were incubated with different concentrations of SB for 1 h followed by stimulation with IL-IF for 12 h. Firefly and Renilla luciferase activities were analyzed in total cell extract. Data are mean ± SD of three different experiments.*, p < 0.001 vs IL-IF. E, Cells pretreated with different concentrations of SB were stimulated with IL-IF under serum-free condition. After 12 h of stimulation, total RNA was isolated and Northern blot analysis for iNOS mRNA was conducted as described under Materials and Methods. F, Cells were treated as in (E) and whole cell lysate was obtained after 24 h which was used to detect protein level of iNOS by Western blotting. Blots were reprobed for Actin. G, Astrocytes were transfected either with 100 nM si-C or si-p38α. After 48 h, whole cell lysate was obtained and level of p38α and actin was detected by Western blotting. H, Cells were transfected either with 100 nM si-C or si-p38α. After 48 h, they were treated with IL-IF for an hour and nuclear extract was prepared, which was used to perform EMSA (Licor Biosciences probe). I, Astrocytes were transfected as in H. After 48 h they were treated with IL-IF for an additional 18 h. Then, whole cell lysate was obtained and was used to detect protein level of iNOS by Western blotting. Blots were reprobed for actin. All results are representative of at least three independent trials.

Ramendra N. Saha, et al. J Immunol. ;179(10):7101-7109.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk