Figure 1.
The CAAX box determines the nature of the posttranslational modification. (A) His-tagged RhoB was purified from Sf9 cell homogenate on Ni-NTA magnetic beads, separated by SDS/PAGE, and analyzed by Western blot with a rabbit RhoB antibody and by silver staining. (B) The isoprenoids linked to His-tagged purified RhoB were determined after methyl iodide cleavage by reverse-phase HPLC. [3H]-Labeled isoprenoids were extracted after methyl iodide treatment and resolved by C18 reverse-phase HPLC as follows: □, Sf9 control; ◊, RhoB-CKVL; ⧫, RhoB-CAIM; ▴, RhoB-CLLL; ●, RhoB-Δ. Isoprenoids were identified by their coelution with isoprenoid standards, such as farnesol (C15) and geranylgeraniol (C20).