Figure 1.
Figure 1. Expression of TLR4-MD2 complex and responsiveness to LPS in DCregs. (A, B) The expression of MHC and costimulatory molecules (A) and the receptors for LPS (B) on DC subsets or macrophages was analyzed by flow cytometry. Data are represented by a histogram in which cells were stained with the indicated mAb (thick lines) or isotype-matched control Ig (thin lines). The results are representative of 4 experiments with similar results. (C) DCs (5 × 105) were stimulated or not stimulated with LPS (1μg/mL) for 6 or 24 hours, and the culture supernatants were analyzed for cytokine production. Data were expressed as mean ± SD of duplicate samples, and the results are representative of 4 experiments with similar results. *P < .01 compared with iDCs by Student paired t test. (D) Cells (5 × 106) were stimulated or not stimulated with LPS (1μg/mL). Western blot shows the cytoplasmic expression of pErk1/2 and pp38, and pIκB-α before and 10 minutes after LPS stimulation, and the nuclear expression of p65 before and 60 minutes after LPS stimulation. The nonphosphorylated kinases and proteins (ERK1/2, p38, and IκB-α) also were analyzed and serve as the control for protein loading. Representative blots from 3 independent experiments are shown. (E) The expression of IκBNS and Bcl-3 in DCs was measured by real-time PCR. Expression of IκBNS or Bcl-3 was normalized to β-actin, and the data were expressed as comparative fold expression of IκBNS or Bcl-3 compared with iDCs. Representative data from 2 independent experiments are shown. *P < .01 compared with iDCs by Student paired t test. (F) DCs (106) were stimulated or not stimulated with LPS (1 μg/mL) for 24 hours, and the concentration of intracellular cAMP was measured. Data were expressed as mean ± SD of duplicate samples, and the results are representative of 3 experiments with similar results. *P < .01 compared with iDCs by Student paired t test. (G) DCs (5 × 105) were stimulated or not stimulated with LPS (1 μg/mL) in the presence or absence of 8-Br-cAMP (200 μM) or Rp-8-Br-cAMPS (1 mM) for 24 hours, and the culture supernatants were analyzed for IL-10 production. Data were expressed as mean ± SD of duplicate samples, and the results are representative of 3 experiments with similar results. *P < .01 compared with LPS stimulation by Student paired t test.

Expression of TLR4-MD2 complex and responsiveness to LPS in DCregs. (A, B) The expression of MHC and costimulatory molecules (A) and the receptors for LPS (B) on DC subsets or macrophages was analyzed by flow cytometry. Data are represented by a histogram in which cells were stained with the indicated mAb (thick lines) or isotype-matched control Ig (thin lines). The results are representative of 4 experiments with similar results. (C) DCs (5 × 105) were stimulated or not stimulated with LPS (1μg/mL) for 6 or 24 hours, and the culture supernatants were analyzed for cytokine production. Data were expressed as mean ± SD of duplicate samples, and the results are representative of 4 experiments with similar results. *P < .01 compared with iDCs by Student paired t test. (D) Cells (5 × 106) were stimulated or not stimulated with LPS (1μg/mL). Western blot shows the cytoplasmic expression of pErk1/2 and pp38, and pIκB-α before and 10 minutes after LPS stimulation, and the nuclear expression of p65 before and 60 minutes after LPS stimulation. The nonphosphorylated kinases and proteins (ERK1/2, p38, and IκB-α) also were analyzed and serve as the control for protein loading. Representative blots from 3 independent experiments are shown. (E) The expression of IκBNS and Bcl-3 in DCs was measured by real-time PCR. Expression of IκBNS or Bcl-3 was normalized to β-actin, and the data were expressed as comparative fold expression of IκBNS or Bcl-3 compared with iDCs. Representative data from 2 independent experiments are shown. *P < .01 compared with iDCs by Student paired t test. (F) DCs (106) were stimulated or not stimulated with LPS (1 μg/mL) for 24 hours, and the concentration of intracellular cAMP was measured. Data were expressed as mean ± SD of duplicate samples, and the results are representative of 3 experiments with similar results. *P < .01 compared with iDCs by Student paired t test. (G) DCs (5 × 105) were stimulated or not stimulated with LPS (1 μg/mL) in the presence or absence of 8-Br-cAMP (200 μM) or Rp-8-Br-cAMPS (1 mM) for 24 hours, and the culture supernatants were analyzed for IL-10 production. Data were expressed as mean ± SD of duplicate samples, and the results are representative of 3 experiments with similar results. *P < .01 compared with LPS stimulation by Student paired t test.

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