Figure 4

MCP110 inhibits Ras/Raf interaction and signaling to ERK in mammalian cells. A. Pulldown assay for Ras-Raf interaction. Pulldowns of active Ras bound to Raf-RBD were done using GST-Raf-RBD as described in Methods. NIH 3T3 cells were treated with vehicle or MCP110 (3, 10 and 30 μM) before and after lysis. Ras was detected by immunoblotting with anti-Ras antibody. Both Raf-RBD-bound Ras (Ras-GTP, upper panel) and total Ras in the lysates (lower panel) are shown. MCP110 disrupted the Ras/Raf interaction in a dose-dependent manner; numbers shown below panels indicate quantitation of Ras pulldown by densitometry, normalized to vehicle control. B. Western blot analysis for active, phospho-ERK1/2. The same lysates from cells depicted in panel A above were immunoblotted for phospho-ERK1/2 (P-ERK, upper panel) and for total ERK1/2 (total ERK, lower panel). Numbers shown below PERK panel indicate quantitation by densitometry, normalized to vehicle control. MCP110 decreased phospho-ERK1/2 in a dose-dependent manner.