Fig. 1

Human MPNST cell lines variably express R-Ras subfamily members. a Lysates from eight human MPNST cell lines probed with antibodies recognizing R-Ras or both R-Ras and R-Ras2 proteins. Blots were probed with an anti-GAPDH antibody to confirm equal loading. Antibody dilutions were: R-Ras 1:10,000, R-Ras plus R-Ras2 (Abnova H000022800_M01) 1:10,000 and GAPDH 1:100,000. b R-Ras and R-Ras2 transcript levels in MPNST lines were quantitated using real-time PCR and compared to the level of expression evident for each transcript in normal human cerebellum tissue, Daoy medulloblastoma cells, MCF-7 breast adenocarcinoma cells, SK-N-MC neuroepithelioma cells, SK-OV-3 ovarian adenocarcinoma cells and U87-MG glioblastoma cells. c Raf-1 RBD pulldown assays performed on ST88-14 cells transiently transfected with plasmids directing the expression of Myc-tagged wild-type R-Ras, Myc-tagged wild-type R-Ras2, HA-epitope tagged dominant negative H-Ras plasmid, HA-epitope tagged dominant negative R-Ras, or eGFP. Only activation of wild-type R-Ras and R-Ras2 is evident in these cells. Upper panel is the material captured with Raf-1 RBD probed with antibodies recognizing the HA or Myc epitope tags on the recombinant proteins. The bottom two panels are clarified lysates probed for the epitope tags or GAPDH, respectively