Figure 13.

The antiapoptotic effect of HO-1 acts via the activation of p38 MAPK. (A) 2F-2B ECs were cotransfected with β-galactosidase, control (pcDNA3), or HO-1 (β-actin/HO-1) expression vectors. Where indicated, ECs were treated with the p38 kinase inhibitor SB203580. Gray bars represent ECs treated with Act.D alone and black bars represent ECs treated with TNF-α plus Act.D. The results shown are the mean ± SD from duplicate wells taken from one representative experiment out of three. (B) BAECs were transfected with a control (pcDNA3) vector and stimulated with TNF-α in the presence (•) or absence (○) of the p38 kinase inhibitor SB203580 (20 μM). MAPK phosphorylation was monitored by Western blot (0, 5, 15, 30, 60, and 120 min after TNF-α stimulation) using antibodies directed against the phosphorylated forms of each MAPK. (C) 2F-2B ECs were cotransfected with β-galactosidase, control, HO-1 (β-actin/HO-1), and where indicated with a phosphorylation-deficient p38/CSBP1 dominant negative mutant (DNM) expression vector. The values indicate the amount of vector used, in nanograms of DNA per 300 × 10³ cells. Apoptosis was induced as in A. Gray bars represent ECs treated with Act.D and black bars represent ECs treated with TNF-α plus Act.D. The results shown are the mean ± SD from duplicate wells taken from one representative experiment out of three. (D) The expression of the p38/CSBP1 dominant negative mutant was confirmed by Western blot using an anti-p38 specific antibody. No Tr., nontransfected ECs.