Inhibition of TNF-–induced MCP-1 mRNA synthesis by p38 inhibitors, SB203580 and SB202190. (A) Total mRNA was extracted from HUVEC exposed to 20 μmol/L SB203580 or SB202190 30 minutes before and during an 8-hour period of stimulation with 2 ng/mL TNF-. Control samples were incubated without p38 inhibitors in the absence or presence of TNF- as indicated. MCP-1 mRNA levels were determined by Northern blot analysis as described in Materials and Methods. Blots were reprobed with an actin cDNA probe to control RNA loading. (B) To assess the effect of p38 inhibition on stability of TNF-–induced MCP-1 mRNA, HUVEC were treated with TNF- in the absence or presence of SB202190 as in (A). After 8 hours (time zero), Act D was added at 2 μg/mL and total RNA isolated at the time intervals indicated (in minutes). Northern blot analysis was performed as described above. (C) Autoradiography bands shown in (B) were analyzed by densitometry and normalized to the RNA content as judged by the levels of 28S rRNA. TNF-–induced MCP-1 mRNA levels at time zero were set as 100%. The decrease of MCP-1 mRNA amounts is shown after exposure to Act D for different time intervals as indicated. Treatment with Act D alone had no effect on the levels of MCP-1 mRNA (data not shown).