In vitro RAS-RAF binding assays

NS1, H-RAS_1–174 and K-RAS_1–174²¹ were expressed in bacteria as HIS tagged fusions using the pHBT vector and purified using nickel agarose beads. Purified RAS was incubated in Loading Buffer (20 mM Tris-HCl pH 7.6, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 4 mM EDTA) with either 2 mM GDP or 2 mM GTPγS for 90 min on ice followed by the addition of 10 mM MgCl₂ to stop loading reaction. GST and GST-RAF RBD were expressed and purified from bacteria as described. For binding reactions, 125 nM RAS was incubated with 125 nM GST or GST-RAF RBD (immobilized on Glutathione Sepharose beads) in the presence or absence of 375 nM NS1 in reaction buffer (25 mM HEPES,pH 7.5, 10% glycerol, 150 nM NaCl, 1%Trition100, 0.25% sodium deoxycholate, 25 mM MgCl₂, 1 mM EDTA) for 30 minutes at 4°C. Beads were then washed 3 times with reaction buffer and bound proteins analyzed by Western blot.