Figure 3
Figure 3. p38 MAPK regulates IL-17 via eIF-4E. (A) CD4 T cells from WT B6 mice were activated under Th17 conditions in the absence (−) or presence (+) of SB203580 (SB) for the indicated periods of time and phosphorylation of eIF-4E at Ser209 (P-eIF-4E) was examined by Western blot analysis. Actin is shown as a loading control throughout. (B) B6 CD4 T cells were activated as in panel A in the presence or absence of BIRB796 (BIRB) for 48 hours, and phosphorylation of eIF-4E at Ser209 was examined by Western blot analysis. (C) CD4 T cells from WT B6 and MKK3−/−MKK6+/− (KO) mice were activated as in panel A and phosphorylation of eIF-4E was examined by Western blot analysis. (D-E) Phosphorylation of eIF-4E in CD4 T cells from WT B10.BR and MKK6-Tg mice before activation (E) or 24 and 48 hours on activation (D) as described in panel A was examined by Western blot analysis. A longer exposure was used for detection of P-eIF-4E in panel E. (F) Relative levels of Il17, c-myc, and Il2 mRNA present in the immunoprecipitates obtained with an anti-eIF-4E Ab or a control IgG using whole-cell lysates from CD4 T cells activated under Th17 conditions for 3 days. Analyses were performed by quantitative real-time PCR and mRNA values for each gene are relative to the levels detected in the control IgG immunoprecipitates. The presence of eIF-4E in the input lysate and the immunoprecipitates was determined by Western blot analysis (right panel). (G) CD4 T cells from WT B6 mice were activated under Th17 conditions in the absence (−) or the presence (+) of the MNK inhibitor CGP57380 (CGP) for the indicated periods of time. Phospho-eIF-4E was examined by Western blot analysis. (H) CD4 T cells from WT B6 mice were differentiated into Th17 cells in the presence of the indicated amounts of CGP57380, and the levels of IL-17 in the culture supernatants after 72 hours were quantified by ELISA. (I) Relative Il17 mRNA levels in CD4 T cells activated as in panel H were assessed after 48 hours by quantitative real-time PCR using β2-microglobulin as the endogenous control. (J) IL-2 production in CD4 T cells activated as in panel H was quantified by ELISA. The significance of differences observed in panels H through J was determined by linear regression analysis (H, P < .0001; I, P = .23; J, P = .27).

p38 MAPK regulates IL-17 via eIF-4E. (A) CD4 T cells from WT B6 mice were activated under Th17 conditions in the absence (−) or presence (+) of SB203580 (SB) for the indicated periods of time and phosphorylation of eIF-4E at Ser209 (P-eIF-4E) was examined by Western blot analysis. Actin is shown as a loading control throughout. (B) B6 CD4 T cells were activated as in panel A in the presence or absence of BIRB796 (BIRB) for 48 hours, and phosphorylation of eIF-4E at Ser209 was examined by Western blot analysis. (C) CD4 T cells from WT B6 and MKK3−/−MKK6+/− (KO) mice were activated as in panel A and phosphorylation of eIF-4E was examined by Western blot analysis. (D-E) Phosphorylation of eIF-4E in CD4 T cells from WT B10.BR and MKK6-Tg mice before activation (E) or 24 and 48 hours on activation (D) as described in panel A was examined by Western blot analysis. A longer exposure was used for detection of P-eIF-4E in panel E. (F) Relative levels of Il17, c-myc, and Il2 mRNA present in the immunoprecipitates obtained with an anti-eIF-4E Ab or a control IgG using whole-cell lysates from CD4 T cells activated under Th17 conditions for 3 days. Analyses were performed by quantitative real-time PCR and mRNA values for each gene are relative to the levels detected in the control IgG immunoprecipitates. The presence of eIF-4E in the input lysate and the immunoprecipitates was determined by Western blot analysis (right panel). (G) CD4 T cells from WT B6 mice were activated under Th17 conditions in the absence (−) or the presence (+) of the MNK inhibitor CGP57380 (CGP) for the indicated periods of time. Phospho-eIF-4E was examined by Western blot analysis. (H) CD4 T cells from WT B6 mice were differentiated into Th17 cells in the presence of the indicated amounts of CGP57380, and the levels of IL-17 in the culture supernatants after 72 hours were quantified by ELISA. (I) Relative Il17 mRNA levels in CD4 T cells activated as in panel H were assessed after 48 hours by quantitative real-time PCR using β2-microglobulin as the endogenous control. (J) IL-2 production in CD4 T cells activated as in panel H was quantified by ELISA. The significance of differences observed in panels H through J was determined by linear regression analysis (H, P < .0001; I, P = .23; J, P = .27).

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