Fig 3.

The dual prenyl-palmitoyl modification is conserved in other CCaX motif proteins. (A) HEK293 cells transfected with FLAG-RalA, FLAG-RalB, or FLAG-PRL-3 were incubated in medium containing 100 μM palmitic acid (PA) or 100 μM 17-ODYA (OD) for 6 h. Lysates were immunoprecipitated with anti-FLAG antibody, and 17-ODYA incorporation was detected with Alexa Fluor 488-azide. (B and C) HEK293 cells transiently expressing FLAG-RalA or its CCaX motif mutants were labeled with 30 μM geranylgeranyl-azide (B) or 100 μM 17-ODYA (C) for 24 h or 6 h, respectively. Geranylgeranylation and palmitoylation were detected with Alexa Fluor 647-alkyne or Alexa Fluor 488-azide, respectively. (D and E) HEK293 cells transiently expressing FLAG-PRL-3 or its CCaX motif mutants were labeled with 30 μM farnesyl-azide (D) or 100 μM 17-ODYA (E) for 24 h or 6 h, respectively. Farnesylation and palmitoylation were detected with Alexa Fluor 647-alkyne or Alexa Fluor 488-azide, respectively.