Figure 1.
Figure 1. TTP regulates the production of IL23 in BMDCs. BMDCs from TTP+/+ or TTP−/− mice were incubated in medium alone or stimulated with 100 ng/ml LPS. (A) Supernatants were collected after 16 h and analyzed for IL23, IL12p70, and IL12/23p40 levels by ELISA. Mean ± SEM of six independent experiments is shown. (B) Total RNA was extracted and analyzed by real-time RT-PCR. One experiment representative of six is shown. (C and D) Spleen cells from TLR4−/− mice were cultured in presence of supernatant from nonstimulated or LPS-stimulated DCs. When indicated, neutralizing antibodies for IL12/23p40, IL23p19, or control IgG (2 µg/ml) were added. After 3 d, supernatants were collected and analyzed for IL17A and IFN-γ levels by ELISA. Mean ± SEM of two experiments performed in triplicate (C) or triplicates from one representative experiment of three (D) is shown. (E) DCs from TTP+/+ or TTP−/− mice were stimulated with 100 ng/ml LPS. After 2 h of stimulation, 10 µg/ml actinomycin d and 1 µM SB202190 were added for the indicated time. Total RNA was extracted and analyzed by real-time RT-PCR. The results represent mean ± SEM of triplicates from one representative experiment of five. (F and G) HEK-293 cells were cotransfected with 400 ng of the indicated reporter plasmid and 100 ng of the indicated expression vector. 24 h after transfection, culture medium was replaced by fresh medium, and 16 h later supernatant were collected and analyzed for IL2 levels by ELISA. Mean ± SEM from three experiments performed in triplicate is shown. (H) Representation of the 3′UTR of the murine IL23p19 mRNA. (I and J) Knockdown experiments were performed by nucleofection of 1 µM Caf1 or control (CTL) siRNA. 24 h later, cells were stimulated as indicated. Total RNA was extracted and analyzed by real-time RT-PCR. The results represent mean ± SEM of triplicates from one representative experiment of two. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

TTP regulates the production of IL23 in BMDCs. BMDCs from TTP+/+ or TTP−/− mice were incubated in medium alone or stimulated with 100 ng/ml LPS. (A) Supernatants were collected after 16 h and analyzed for IL23, IL12p70, and IL12/23p40 levels by ELISA. Mean ± SEM of six independent experiments is shown. (B) Total RNA was extracted and analyzed by real-time RT-PCR. One experiment representative of six is shown. (C and D) Spleen cells from TLR4−/− mice were cultured in presence of supernatant from nonstimulated or LPS-stimulated DCs. When indicated, neutralizing antibodies for IL12/23p40, IL23p19, or control IgG (2 µg/ml) were added. After 3 d, supernatants were collected and analyzed for IL17A and IFN-γ levels by ELISA. Mean ± SEM of two experiments performed in triplicate (C) or triplicates from one representative experiment of three (D) is shown. (E) DCs from TTP+/+ or TTP−/− mice were stimulated with 100 ng/ml LPS. After 2 h of stimulation, 10 µg/ml actinomycin d and 1 µM SB202190 were added for the indicated time. Total RNA was extracted and analyzed by real-time RT-PCR. The results represent mean ± SEM of triplicates from one representative experiment of five. (F and G) HEK-293 cells were cotransfected with 400 ng of the indicated reporter plasmid and 100 ng of the indicated expression vector. 24 h after transfection, culture medium was replaced by fresh medium, and 16 h later supernatant were collected and analyzed for IL2 levels by ELISA. Mean ± SEM from three experiments performed in triplicate is shown. (H) Representation of the 3′UTR of the murine IL23p19 mRNA. (I and J) Knockdown experiments were performed by nucleofection of 1 µM Caf1 or control (CTL) siRNA. 24 h later, cells were stimulated as indicated. Total RNA was extracted and analyzed by real-time RT-PCR. The results represent mean ± SEM of triplicates from one representative experiment of two. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

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