FIG. 6.

MAPK p38 activity is required for the expression of TTP in response to LPS. (A) RAW264.7 cells preincubated for 15 min with vehicle or 1 μM SB203580 and then stimulated with LPS for the times indicated. Cytoplasmic extracts were prepared and separated by SDS-polyacrylamide gel electrophoresis on a 10% gel and then analyzed by Western blotting using an anti-N-terminal TTP antiserum. The letters at the right (a, b, and c) indicate TTP bands of differing mobility. The positions of molecular size markers are indicated at the left of the gel. (B) RAW264.7 cells preincubated for 15 min with vehicle or 0.1 to 10 μM SB203580, as indicated, and then stimulated with 10 ng of LPS per ml for 2 h. Cytoplasmic extracts were prepared and used in EMSAs with 20 fmol of a full-length human TNF-α 3′ UTR probe. (C) RAW264.7 cells treated as described for panel B, with RNA then extracted and subjected to Northern blotting using a 1-kb TTP cDNA probe. As a loading control, 28S rRNA was visualized by staining of the gel with Sybr green prior to transfer and Northern blotting.