Fig. 4. Activation of MAP kinase by IGF-I. Nonmalignant and malignant mammary cell lines were grown to confluence, serum-starved overnight, and treated with IGF-I for 10, 30, or 360 min except for BT-474 and BT-20, which were incubated for 15, 30, 45, 90, or 180 min. Unstimulated cells were mock-treated without IGF-I for 30 min. Twenty µg of each RIPA-extracted cell lysate were fractionated by SDS-PAGE and immunoblotted with anti-phosphoERK1/ERK2. Arrows, the phosphorylated forms of p44 (ERK1) and p42 (ERK2) MAP kinase. Although ERK2 phosphorylation in AB589 cells was detected with ECL exposures >2 min, ERK1 phosphorylation in these cells was not detectable at any exposure up to 25 min. Exposure times of <30 s were required to detect phosphorylation in the other cell lines. *, nonmalignant.