fig.s1. effects of various inhibitors on lps-induced tnfα mrna expression in raw264.7 cells. the...

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Fig.S1. Effects of various inhibitors on LPS-induced TNFα mRNA expression in RAW264.7 cells. The same experimental procedures were performed as described in Figure 1 A and B. The level of TNFα mRNA was determined by using specific primers for TNFα (5’-GACCCTCACACTCAGATCATCTTCT-3’ and 5’-CCTCCACTTGGTGGTTTGCT-3’).

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Fig.S2. The inhibitory effect of 1 mM of SB203580 on LPS-induced TTP mRNA expression. RAW264.7 cells were pretreated with 1 mM of SB203580 for 30 min, and then followed by treated with LPS for 30 min or 2 h. The total RNAs were isolated for real-time PCR analysis by using specific primers for TTP .

Fig.S3 Induction of TTP expression by recombinant TNFα treatment in NIH3T3 cells. NIH3T3 cells were pretreated or untreated with 20 mM BAY for 30 min followed by treated with 10 ng/ml recombinant TNFα for 0, 15 min, 30 min and 60 min and the whole cell extracts were isolated for Western blotting analysis by using anti-TTP antibody. Anti-α-tubulin were used as internal control.

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Fig.S4. The effect of MG132 on LPS-induced TTP protein expression. RAW264.7 cells were pretreated with 10 mM of MG132 for 1 hr (Lanes 1, 4, and 7) or 20 mM of MG132 for 30 min (Lanes 2, 5, and 8), and then followed by treated with 100 ng/ml of LPS for 30 min (Lanes 3-5) or 2 h (Lanes 6-8). The cytosolic extracts were isolated for Western blotting with anti-TTP and anti-tubulin antibodies.

Fig.S5. The activity of intron 1-containing TTP promoter. The TTP promoter spanning from -1026 to +759 was PCR cloned by primers : 5’-AGTCTGACATTGAACGCCTG-3’ and 5’-GAGGAACAGGGTTCGGTTAG-3’, indicated as TTP (intron 1). RAW264.7 cells were co-transfected with TTP (intron 1)-driven luciferase repoter and CMV-driven -galactosidase plasmids. 24 h after trasfection, the cells were treated with 100 ng/ml of LPS together with or without 20 μM of BAY for 2 h. The cells were harvested for luciferase and -galactosidzse analysis. The presented luciferase activity was normalized with -galactosidase activity. This experiment was repeated twice independently. 

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