FIG. 5.

p75-Ras-GRF1 is required for the activation of Ras in response to c-Jun/AP-1. (A) p75-Ras-GRF1 and Ras interaction was determined by pull-down experiments with GST-Ras15A-agarose beads. The pulled-down complexes were separated by SDS-PAGE and immunoblotted with antibodies against Ras-GRF1 and GST. Lanes 1 and 2 represent control (without doxycycline) and c-Jun-containing (with doxycycline) extracts incubated with GST-Ras15A-agarose beads, and lane 3 shows p-75-Ras-GRF1 in the total protein lysate. Lane 4 represents c-Jun-containing extracts incubated with GST-agarose beads as a negative control. The top panel shows immunoblots for Ras-GRF1, and the bottom panel shows immunoblots for GST. (B and C) GTP-bound Ras was extracted from control (without doxycycline) and c-Jun-expressing (with doxycycline) Rat1a-c-Jun4 cells grown under adherent (B) and anchorage-independent (C) conditions with Raf-1-RDB agarose beads. The complexes were immunoblotted with a Ras monoclonal antibody. Equal amounts of total Ras were present in control and doxycycline-treated cell lysates. (D) Activated Ras pull down assays using protein extracts from Rat1a-c-Jun4 cells grown in suspension for 48 h with and without doxycycline in the presence of 10 μM sense or antisense Ras-GRF1 oligomers. Immunoblots with Ras antibody are shown. The results are representative of triplicate experiments.