Figure 6.

The activities of the Ras–Raf-1–ERK pathway, p38-MAPK, and p46/p54–JNK1 were examined in Tg-DN-Trx1 mice and NTg littermates. (a) Some mice were subjected to aortic banding for 2 weeks. Activities of p42/p44–ERK (a), Raf-1 (b), p38-MAPK (e), and p46/p54–JNK1 (f) were determined using anti-phosphospecific Ab’s. The same filter was reprobed with respective non-phosphospecific Ab. (e) Cell extracts prepared from cardiac myocytes overexpressing mammalian sterile 20–like kinase 1 (Mst1) were used as positive control (P/C). The gel picture is representative of three to ten experiments in each immunoblot analysis. (c) The activity of Ras was determined using the Ras-binding domain in Raf-1 coupled with agarose. (d) S-thiolation of Ras was determined as described in Methods. COS-7 cells grown in 60-mm dishes were transfected with the indicated expression plasmids (2 μg). Cells were incubated with biotinylated cysteine (0.5 mM), and S-thiolated proteins were isolated by streptavidin-Sepharose. Samples were subjected to immunoblot analyses with anti-Ras Ab. AS-Trx1, antisense Trx1.