Fig 2.

The CCaX motif is modified with a prenyl group and a palmitate on the same molecule. Sf9 cells expressing His-H-Ras (A) or His-bCdc42 (B) and its CCaX motif mutants (C) were incubated in medium containing 30 μM farnesyl-azide (Far-azide) or geranylgeranyl-azide (GG-azide) for 24 h. Lysates were incubated with Ni-NTA agarose, and His-tagged proteins were eluted from the resin. Then, hydroxylamine (NH₂OH)-sensitive palmitoylated proteins were specifically immobilized on thiopropyl-Sepharose resin using acyl-RAC, and prenylation of captured proteins was detected with Alexa Fluor 647-alkyne using click chemistry. Dually prenylated and palmitoylated H-Ras (Far-Palm H-Ras) or bCdc42 (GG-Palm bCdc42) was analyzed by in-gel fluorescence.