Activation of endothelial p38 MAP kinase by TNF-α is efficiently inhibited by SB202190. To confirm efficient inhibition of p38 activity in endothelial cells to be studied by microarray profiling, immune complex kinase assays (A) and flow cytometry (B) were performed on cells obtained from parallel experiments. (A) HUVECs were exposed to medium or TNF-α for 30 or 300 minutes in the absence or presence of 10 μM SB202190, which had been added to the culture medium 30 minutes prior to stimulation. After cell lysis, MK-3/3pK, a kinase acting downstream of p38, was immunoprecipitated and assayed for activity using Hsp27 as substrate. Protein loads were controlled by Western blot using an antiserum against MK-3/3pK. (B) Cells were stimulated with TNF-α for 5 hours in the absence or presence of 10 μM SB202190 as indicated and subsequently studied by flow cytometry for surface expression of an established p38-dependent gene, VCAM-1 (thick lines). Isotype controls are additionally shown (thin lines).