Contact between RAMOS-wt and stromal cells does not change the phosphotyrosine profile of RAMOS-wt, but enhances Elk1-bound kinase activity. Stromal cells and RAMOS-wt cells were incubated at 37°C, collected, lysed, electrophoresed, and immunoblotted as detailed in the Materials and Methods. (A) Stromal cells in medium for 5 minutes (−) or in contact with RAMOS-wt for the times indicated (in minutes). (B) RAMOS-wt cells in medium (−) or in contact with stromal cells for the times indicated (in minutes). (Upper panel) Antiphosphotyrosine immunostaining. (Middle panel) Anti-vav immunostaining. (Lower panel) Anti–β-tubulin immunostaining. Asterisks indicate the major stromal cell protein species demonstrating consistent increases in tyrosine phosphorylation. (C) RAMOS cells were incubated in medium alone (cont), on stromal cells (stroma), or with 20 ng/mL phorbol 12-myristate 13-acetate (PMA) for 5 minutes at 37°C. Lysates were assayed by solid-phase kinase assay (γ32P-GST-Elk1, 3.5 hours of exposure, upper panel) or by immunostaining with anti-ERK2 (lower panel) as detailed in the Materials and Methods. Data in (C) were obtained in the same experiment as Fig 2C. Results are representative of three (A and B) and two (C) individual experiments.
