Figure 2.

Cellular levels of the active, GTP-bound forms of germline KRAS mutants. Pull-down experiments of GTP-bound KRAS proteins (RAS_GTP) were performed in COS-7 cells transiently expressing either KRAS^wt or germline KRAS mutants in the presence (A) and in the absence of serum (B). Irrespective of culture conditions almost all KRAS mutants showed an increased GTP-bound level. Purified RAS-GAP, which was added to the cleared cell lysates proved the GAP sensitivity of the mutants (B, middle panel). GAP resistant mutants, RAS^P34L, RAS^P34R, and RAS^G60R, resided in the active state comparable to oncogenic RAS^G12V. Total amounts of recombinant RAS are shown for equal expression and loading. Anti-RAS antibodies used in these experiments were anti-RAS (RAS10 clone, Upstate-Millipore™, mutants p.G60R, p.D153V, p.F156L) and anti-RAS (BD Transduction Laboratories™, wt and all other mutants), because some mutations modified the RAS epitope recognized by the respective antibodies. Additional information is given in Supp. Fig. S2.