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Blood, Vol. 112, Issue 3, 635-643, August 1, 2008
Mammalian target of rapamycin and glycogen synthase kinase 3 differentially regulate lipopolysaccharide-induced interleukin-12 production in dendritic cells
Blood Ohtani et al.
112: 635
Supplemental materials for: Ohtani et al
Files in this Data Supplement:
- Figure S1. The signal profiles of p70S6K in LPS-stimulated BMDCs (JPG, 56.2 KB)
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The quantitative analysis of signal intensity of the p70S6K bands in Figure 2 along a vertical axis from bottom to top.
- Figure S2. The effect of rapamycin on LPS-induced IL-12 and IL-10 productions in human MDDCs (JPG, 24.7 KB)
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Human MDDCs were stimulated with 10 µg/ml LPS in the presence or absence of 100 ng/ml rapamycin for 20 h, and assayed for the production of IL-12p70 and IL-10 by ELISA. Similar results were obtained with three independent blood donors.
- Figure S3. FK506 blocks the effect of rapamycin on cytokine production and p70S6K phosphorylation (JPG, 71.1 KB)
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BMDCs were pretreated with or without 10 ng/ml rapamycin along with the indicated concentrations of FK506 for 30 min or left untreated before being stimulated with 1 µg/ml LPS for 1h (for Western blot) or 18 h (for ELISA). The production of IL-12p70 and IL-10 in culture supernatants was assayed by ELISA (upper panel). Data are indicated as median ± SD of duplicate samples. The cell lysates were analyzed for p70S6K by Western blotting (lower panel). Note that the mobility shifts of p70S6K caused by multiple phosphorylation are shown. The quantitative data of signal intensity of p70S6K band along a vertical axis from bottom to top are indicated below.
- Figure S4. The signal profiles of p70S6K in BMDCs expressing Rheb Q64L and mock-infected BMDCs (JPG, 45.6 KB)
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The quantitative analysis of signal intensity of p70S6K band in Figure 4A along a vertical axis from bottom to top.
- Figure S5. The effect of rapamycin on LPS-induced phosphorylation of STAT3 in human MDDCs (JPG, 49.9 KB)
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Human MDDCs were pretreated with or without 100 ng/ml rapamycin for 20 min, and then stimulated with 10 µg/ml LPS for the indicated times. The cell lysates were analyzed for phospho-STAT3, STAT3, p70S6K and ERK2 by Western blotting. The white lines indicate that intervening lanes have been spliced out. The right panel indicates the relative intensities of phosphorylated STAT3 normalized by STAT3 signals.
- Figure S6. The effect of exogenous IL-10 on LPS-induced IL-12 production in IL-10−/− BMDCs (JPG, 44 KB)
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BMDCs from IL-10−/− mice were pretreated with or without 10 ng/ml rapamycin for 20 min before being stimulated with 1 µg/ml LPS along with the indicated concentrations of IL-10 for 24 h, and assayed for the production of IL-12p70 by ELISA. Data are indicated as median ± SD of duplicate samples.
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