The proteasome inhibitor ALLN, the antioxidant PDTC, or NaSal, but not dexamethasone, decreases constitutive and IL-4–induced expression of mRNA for P-selectin and VCAM-1 in HUVEC. (A) Confluent HUVEC in 25-cm² flasks were preincubated for 1 hour at 37°C in the presence or absence of 25 μmol/L ALLN, 50 μmol/L PDTC, 10 mmol/L NaSal, or 1 μmol/L dexamethasone (Dex). The cells were then incubated for 20 hours at 37°C with or without 10 ng/mL of IL-4 in the continued presence of the respective pharmacological agent. Total RNA was then isolated, and Northern blot analysis was performed with sequential hybridization of the membrane with cDNA probes for P-selectin, VCAM-1, or CHO-B. (B) The relative levels of P-selectin mRNA were quantified by densitometric scanning, normalized according to the level of CHO-B mRNA, which was not affected by IL-4. (C and D) The experiment was performed exactly as in panels A and B, except that cells were incubated with the following controls: EtOH used as the diluent for dexamethasone, DMSO used as the diluent for ALLN, 25 μmol/L Indo, or 25 μmol/L of ALLM, a protease inhibitor with relatively little activity against the proteasome. The data in panels A and B are representative of five experiments. The data in panels C and D are representative of two experiments. The data for IL-4–stimulated cells in panel B were also pooled with the analogous results from the other four experiments. Statistical analysis of the pooled results revealed that ALLN, PDTC, or NaSal significantly decreased IL-4–induced P-selectin mRNA levels (P ≤ .01).