Stimulation of HUVEC with TNF- plus OSM does not cooperatively increase mRNA for P- and E-selectin. (A) Confluent monolayers of HUVEC were treated with 10 ng/mL human OSM, 100 U/mL human TNF-, or a combination of both cytokines. After the indicated time, total RNA was isolated, and 20 μg of RNA was electrophoresed and then transferred to a membrane for Northern blot analysis. The same membrane was sequentially hybridized with the indicated cDNA probes. The mobilities of the hybridized transcripts corresponded to published values. (B) HUVEC were treated with 10 ng/mL OSM in the presence or absence of 10 μg/mL cycloheximide. After the indicated time, total RNA was isolated and analyzed by Northern blotting. (C) HUVEC were treated with 1 μg/mL OSM or 100 U/mL TNF- in the presence or absence of 5 μg/mL actinomycin D. After 4 hours, the cells were lysed and analyzed by Western blotting with a MoAb to E-selectin.