Protein Signaling Pathway Analysis

GFP (sc-9996), Raf (sc-133, sc-227) antibodies and purified recombinant MEK-1 (sc-4025) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). AKT (AH01112) and Phospho-AKT (44623G) were from Invitrogen (Waltham, MA, USA). p42/44 MAPK (ERK1/2), pp44/42 MAPK (ERK T202/Y204), ppMEK (S217/221) and pAKT (S473) antibodies were from Cell Signaling Technology (Danvers, MA, USA). Fluorophore-conjugated secondary antibodies were from LI-COR Biosciences (Lincoln, NE, USA). LY294002 was purchased from LC laboratories (Woburn, MA, USA). U0126 was purchased from Alfa Aesar (Ward Hill, MA, USA). Restriction endonucleases were obtained from New England Biolabs (Ipswich, MA, USA). pEGFP-C1 was from Clontech Laboratories (Mountain View, CA, USA). GFP-R-Ras constructs were made as described in Wurtzel et al.^{12 (link)} GFP-H-Ras G12V was a gift from K Svoboda (Addgene plasmid 18666). GFP-H-Ras (1–174)G12V- R-Ras (204–218) was generated from GFP-H-Ras G12V by PCR. GFP-RNex-H-Ras was generated by insertion of the first 26 amino acids of R-Ras from an R-Ras N-terminal domain construct originally described in Silver et al.,^{67 (link)} into the GFP-H-Ras background.