Extended Data Fig. 2: Association of KRAS4A with HK1 and HK2 requires prenylation but is diminished by palmitoylation.

a, HeLa cells expressing the indicated, Flag-tagged GTPase were lysed, KRAS4A or RAC1 were immunoprecipitated and the precipitates were blotted for Flag-tagged GTPases or endogenous HK1 or HK2. b, To confirm that the results in a reflect membrane targeting rather than GTP loading, the relative GTP loading of the KRAS4A proteins was determined by GST–RAF1-RBD affinity capture. Left, Flag–KRAS4A with an activating G12V mutation and either a native (N) membrane-targeting sequence or one with the indicated substitution were expressed in HEK293 cells. Right, in addition, cells expressing Flag–KRAS4A(G12V) were treated with 2-BP to inhibit palmitoylation or FTI to inhibit farnesylation. The total level of Flag–KRAS(G12V) was measured by anti-Flag immunoblot of 1% of the lysate (bottom) and GTP-bound Flag–KRAS4A was measured by affinity purification of the remaining lysate with GST–RAF1-RBD (top). The number under each lane is the amount of GTP-bound KRAS4A relative to lane 1 after normalization for expression (bottom). The immunoblots shown are representative of two independent experiments (a, b).