Analysis of the MAPK-pathway in betulinic acid-treated cells. (A) Upper panel: Effect of PD98059 (10 µg/ml) on Mcl-1 expression in the presence or absence of betulinic acid (10 µg/ml) measured after 18 h. Lower panel: immunoblot analysis of the same total cellular lysates with an antibody specific for ERK1 and —2. Arrows indicate the positions of the 42 and 44 kDa isoforms. (B) Simultaneous phospho-MEK1/2 and phospho-MAPK analysis of a vector control (Ctrl) and activated mutant N-ras (N) transfected melanoma cell line (518A2). Analysis was performed after 36 h serum starvation of cells. After starvation, 10 µg/ml betulinic acid was added for the indicated times (0 and 6 h incubation). Arrows identify individual protein bands.