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1.
FIGURE 7

FIGURE 7. From: Dynamic Protein Associations Define Two Phases of IL-1β Transcriptional Activation.

NF-κB/IL-1β associations are independent of IRF protein association. A and B, Time course of the NF-κB p65 subunit associating with the IL-1β promoter (A) or enhancer (B) in murine bone marrow-derived macrophages stimulated with LPS as indicated, then analyzed by ChIP. Shown are averages and SE from three independent assays. C, Effect of inhibiting TRAF6/NF-κB activation on IRF protein association with the IL-1β enhancer as measured by ChIP. MM6 monocytes were pretreated with a TRAF6 inhibitor peptide that blocks NF-κB activation, then treated as shown before analysis of IRF protein association with the proximal IL-1β enhancer (EIV). Shown are averages and SE of four independent determinations.

Yue Zhang, et al. J Immunol. ;181(1):503-512.
2.
FIGURE 4

FIGURE 4. From: Dynamic Protein Associations Define Two Phases of IL-1β Transcriptional Activation.

CK2 inhibition blocks NF-κB activation by two measurements. A, Western blots showing IκB-α levels in MM6 cells stimulated for the indicated times in the absence (lanes 1–4 and 9–12) or presence (lanes 5–8 and 13–16) of the CK2 inhibitor apigenin and the absence (lanes 1–8)or presence (lanes 9–16) of the proteosome inhibitor MG132. B, Apigenin blocks NF-κB p65 phosphorylation. Western blots showing phospho-p65 (Ser276) in MM6 cells stimulated with LPS for the indicated time (lanes 1–9) in the presence of the apigenin diluent (DMSO; lanes 4–6) or apigenin (lanes 7–9). Bottom panel shows similar loading as measured by calnexin levels. Each panel shows one of three to four similar results.

Yue Zhang, et al. J Immunol. ;181(1):503-512.
3.
FIGURE 5

FIGURE 5. From: Dynamic Protein Associations Define Two Phases of IL-1β Transcriptional Activation.

Mutated PU.1 expression reveals two mechanistic phases of inducible IL-1β transcription. A, PU.1 mutated serine-to-alanine at amino acid 148 was expressed in monocytes, then successfully transfected cells were isolated and stimulated for times indicated on the x-axis. Shown are IL-1β mRNA levels normalized to β2-microglobulin, expressed relative to mRNA in unstimulated cells, using the Δ(ΔCt) method as detailed in . Black line shows IL-1β mRNA in mutated PU.1-expressing cells; gray line shows IL-1β mRNA in control (GFP) transfected cells. Bars are average and SE from five independent determinations. B, Total levels of PU.1 mRNA are similar in mock (■) and mutated PU.1-expressing cells (□) analyzed by real-time RT-PCR as in .

Yue Zhang, et al. J Immunol. ;181(1):503-512.
4.
FIGURE 6

FIGURE 6. From: Dynamic Protein Associations Define Two Phases of IL-1β Transcriptional Activation.

Time course of IRF protein expression and IRF/IL-1β enhancer association in LPS-stimulated MM6 monocytes confirms two mechanistic phases of inducible IL-1β production. A, Monocytes were stimulated in the absence or presence of the CK2 inhibitor apigenin for the indicated times. IRF protein was measured on Western blots using anti-IRF-4 (top) or anti-IRF-8 (bottom). The calnexin loading control for each IRF blot is shown immediately beneath it. Shown are representative of three independent determinations. B, Association of IRF-4 (middle bars) or IRF-8 (rightmost bars) with the IL-1β enhancer in monocytes stimulated for times indicated at right. Shown are the averages and SE from 3 to 11 independent determinations. IRF-4 and IRF-8 association in unstimulated cells is significantly different as analyzed by Student's t test (p = 0.0076), but is similar 60 min poststimulation (p = 0.49).

Yue Zhang, et al. J Immunol. ;181(1):503-512.
5.
FIGURE 3

FIGURE 3. From: Dynamic Protein Associations Define Two Phases of IL-1β Transcriptional Activation.

CK2 regulates IL-1β transcription through effects on IRF-4/enhancer association in MM6 monocytes. A, Monocytes were treated with inhibitors indicated at right before addition of the LPS for 1 h. IL-1β mRNA was quantified as in . B and C, Effect of the kinase inhibitors apigenin and SB203580 (inhibiting CK2 or p38, respectively) on IL-1β promoter accessibility as measured by CHART-PCR. Promoter regions centered ~100 bp (p(−I), B) or 400 bp (p(−V), C) upstream of transcription start were analyzed. Shown are averages and SD of three independent experiments. Units of MNase were determined independently of units used in . D, Normalized mRNA levels for CK2 (left) and IL-1β (right) for monocytes treated with LPS for the indicated time quantified as in . E, Association of IRF-4 to the IL-1β enhancer in monocytes treated as indicated at right then assayed by chromatin immunoprecipitation. Cells were pretreated with inhibitor for 3 h (apigenin) or 10 min (SB203580) before stimulation with LPS for 1 h. Real-time PCR results show averages and SD from three experiments. F, Positive control for apigenin-mediated CK2 inhibition in MM6 cells. Two independent preparations of stimulated MM6 cells were tested for CK2 activity in the presence or absence of apigenin. Bars show average of duplicate measurements from each preparation.

Yue Zhang, et al. J Immunol. ;181(1):503-512.
6.
FIGURE 2

FIGURE 2. From: Dynamic Protein Associations Define Two Phases of IL-1β Transcriptional Activation.

Multiple transcription factors constitutively and inducibly associate with IL-1β regulatory regions. Transcription factor association with the IL-1β promoter or enhancer was measured by ChIP of unstimulated (■) or stimulated (□; 100 ng/ml E. coli LPS for 2 h) MM6 monocytes. A, Association of TBP, SSRP, or c-Jun with the IL-1β promoter as labeled along the x-axis. B, Association of TBP, c-Jun, and c-fos to the transcription start site proximal (left bars) or distal (right bars) enhancer as measured by amplicons centered at −2907 or −3155, respectively. Both regions were assayed based on previous descriptions of proximal and distal AP-1 sites in the enhancer. C, IRF association with the promoter proximal IL-1β enhancer region, which contains a composite PU.1/IRF site. Cells were stimulated for 1 h for this panel only. Differences between signals in unstimulated vs stimulated anti-IRF-4- but not anti-IRF-8-precipitated DNAs were statistically significant (p = 0.046 or p > 0.1 by Student's t test, respectively). Most ChIP assays were completed three to seven times; TBP/enhancer association was completed twice. Shown are averages and SE of association except for TBP/enhancer ChIPs, which show averages and range of points. All ChIP values for transcription factor-specific precipitations are normalized to PCR signal from samples precipitated with a control anti-histidine tag Ab, whose value was set to 1.

Yue Zhang, et al. J Immunol. ;181(1):503-512.
7.
FIGURE 1

FIGURE 1. From: Dynamic Protein Associations Define Two Phases of IL-1β Transcriptional Activation.

The IL-1β promoter is packaged into an accessible structure in primary human monocytes but not in B cells. Nuclei from primary cells as indicated were digested with MNase, and chromatin accessibility was measured by real-time PCR (CHART-PCR) using primers that amplify DNA centered at 100 (p(−I)) or 400 (p(−V)) bp upstream of the IL-1β transcription start site. A, IL-1β promoter accessibility at two regions in human peripheral blood (PB) monocytes measured after treatment with the indicated units of MNase. B, IL-1β promoter accessibility to 2 units MNase in PB B cells from healthy human donors, tonsil B cells, 293 human kidney cells, or Jurkat T cells. C, IL-1β mRNA levels in PB B cells, MM6 monocytes, or primary human monocytes (as indicated) stimulated with LPS. IL-1β and β2-microglobulin (the normalization control) mRNA were quantified by real-time RT-PCR. Fold stimulation relative to unstimulated cells calculated by the Δ(ΔCt) method is shown. IL-1β transcript levels in B cells from three donors are shown along with one representative mRNA time course from the monocyte cell lines and primary monocytes, both of which recapitulate numerous published IL-1β mRNA profiles. D, Accessibility of the IL-1β promoter in the pro-B cell line 1–2, or murine bone marrow pro-B and pre-B cells, assayed as in A. E, Accessibility of the IL-1β promoter ~400 bp upstream of transcription start in B cell precursors as outlined in A. A and D show averages and SD from three independent determinations. B and E show averages and ranges from two independent experiments.

Yue Zhang, et al. J Immunol. ;181(1):503-512.

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