Levels of activated MAP kinase, IGF-1 receptors, and IGF-1 receptor mRNA in ELM cells grown long-term on stroma or in SCF or IGF-1. (A) Phosphorylation status of MAP kinase (ERK1) in three IGF-1–selected clones (20.1b, 20.2b, and 20.2g) or two stroma-independent variants isolated previously.16 Cells were starved in 0.5% serum medium for 6 hours before analysis. As a positive control, serum-starved ELM cells were stimulated with 20% serum for 10 minutes. Activated ERK1 and ERK2 were determined by Western blotting with antibodies against the activated form of MAP kinase (top panel) or with antibody against total MAP kinase as a loading control (bottom panel). (B) Levels of IGF-1 receptors in ELM clones isolated by long-term growth in IGF-1, determined by Western blotting. (C) Numbers and affinities of IGF receptors in ELM clones selected by long-term growth in SCF or IGF-1, compared with ELM cells growing on stroma, as determined by IGF-1–binding studies. Top panel, typical Scatchard plots for ELM cells freshly removed from stroma, and two ELM clones maintained long-term in either IGF-1 (20.2B) or SCF (20.1D). Bottom panel, average numbers of receptors/cell and affinities (K^d) calculated from the Scatchard plots of two separate experiments. (D) Levels of IGF mRNA by RT-PCR.