Generation of lck-MKK3b(A)/MKK6b(A) transgenic mice.

(Panel A) Schematic representation of the MKK3b(A)/6b(A) transgene. MKK3b(A) and MKK6b(A) were subcloned between the proximal lck promoter and human growth hormone (hGH) polyadenylation signals and intron sequence. Arrowheads indicate the locations of the transgenic-specific PCR primers. (Panel B) RT-PCR analysis of MKK3b(A) and MKK6b(A) transgene expression in peripheral lymphocytes. mRNA prepared from peripheral blood lymphocytes of 5-week-old MKK3b/6b(A) transgenic mice and normal littermate control (NLC) was amplified using the transgenic-specific PCR primers. The PCR products were resolved using agarose gels. (Panel C) Expression of MKK3b(A) and MKK6b(A) proteins in transgenic mice. The expression levels of MKK3 and MKK6 protein in thymocytes from MKK3b(A)/6b(A) transgenic mice and NLC mice were determined by immunoblots. (Panel D) T-cell activation stimulates mainly p38α. Thymocytes were rested for 4 hours after isolation from mice and then stimulated with immobilized anti-CD3 (10 μg/mL) plus anti-CD28 (2.5 μg/mL) or hydrogen peroxide (500 ng/mL). Lysates were prepared 20 minutes after treatments, and the kinase activity of p38α and p38δ isolated by immunoprecipitation was determined using GST-ATF-2 (1-109) as substrate. (Panel E) Inhibition of p38α activation in different lck-MKK3b(A)/MKK6b(A) transgenic mice. Rested thymocytes from NLC, line-9, and line-2 transgenic mice were stimulated with immobilized anti-CD3 (10 μg/mL) and anti-CD28 (2.5 μg/mL) for 20 minutes and 60 minutes, respectively, and cell extracts were prepared. The contents of the phosphorylated p38 MAPK (pp38) and the total p38α MAPK were determined by immunoblots using anti–phosphorylated T180/Y182 p38 MAPK antibody (Cell Signaling) and anti–p38α C-20 (Santa Cruz Biotechnology), respectively. Activation of ERK was used as an internal control using anti–phosphorylated T202/Y204 ERK (Cell Signaling).