Contact with RAMOS-wt or RAMOS-4c increases phosphorylation of stromal cell ERK2. Stromal cells were incubated in medium alone (−) or in contact with RAMOS-wt (wt) or RAMOS-4c (4c) for 5 minutes at 37°C. Stromal cells and RAMOS cells were collected separately and lysed as detailed in the Materials and Methods. (A and B) Lysates were incubated with antibody-coated protein A sepharose overnight at 4°C. Sepharose beads were sedimented by centrifugation for 10 seconds. The supernatant lysate was removed from selected samples and then immunoprecipitates were washed, eluted, and electrophoresed. (A) Anti-ERK2 immunoprecipitates. RAMOS represents immunoprecipitation of a pool of equal amounts of RAMOS-wt and RAMOS-4c lysate. (B) Supernatant lysates removed from rabbit antimouse IgG (cont) or anti-ERK2 immunoprecipitations of stromal cells after RAMOS-wt contact. (Upper panels) Antiphosphotyrosine immunoblotting. (Lower panels) Anti-ERK2 immunoblotting. (C) Lane 1 (cont) represents control glutathione sepharose (lacking GST-Elk1) incubated with stromal cell lysate. Lanes 2 through 4 represent lysate from stromal cells, stromal cells in contact with RAMOS-wt, and stromal cells in contact with RAMOS-4c subjected to solid-phase kinase assay using GST-Elk1 as a substrate (upper panel) or immunostained with anti-ERK2 (lower panel). For lanes 5 and 6, lysates were precleared by immunoprecipitation with anti-ERK2 (*) before incubation with GST-Elk-1-sepharose. Lane 7 represents Elk1-GST sepharose not incubated with lysate. Autoradiography of γ32P-GST-Elk1 represents 10 minutes of exposure. Results are representative of three (A and B) and two (C) individual experiments.
