Figure 3

RocA disrupts the RAS-ERK pathway by targeting the CRaf-PHB interaction in AsPC-1 cells. A. Chemical structure of RocA. B. AsPC-1 cells were treated with RocA at various concentrations or time periods as indicated and then the activation status of ERK was examined by immunoblot analysis. ERK1 and α-tubulin were used as controls. C. Analysis of PHB-CRAF membrane localization in AsPC-1 cells in the presence of RocA or DMSO. AsPC-1 cells were treated with RocA (100 nM) or DMSO for 16 h. Whole cell lysates as well as cell membrane and cytosol fractions were prepared and then subjected to immunoblot analysis with the indicated antibodies. Phospho-CRAF (pSer338) was used as marker of activity and α-tubulin as the loading control. D. Immunoprecipitation of PHB protein and associated CRAF from AsPC-1 cells following 4 h of treatment with RocA (100 nM) or DMSO. E. Confocal microscopic images of the localization of PHB (red) and p-ERK1/2 (green). AsPC-1 cells were treated with RocA (100 nM) or DMSO for 4 h and then stimulated with EGF (50 ng/ml) for 15 min. AsPC-1 cells were incubated with antibodies specific for PHB or p-ERK1/2. Nuclei were counterstained with DAPI. Scale bars, 10 μm. Results are representative of three independent experiments.