Fig. 10. Myristoylation of hARL5. (A) Recombinant hARL5, hARL1, and yARL3 were synthesized in E. coli co-expressing yeast N-myristoyltransferase that were grown in medium containing (³H)-myristic acid. Samples of bacterial proteins (20 µg) were subjected to SDS-PAGE in a 15% gel, which was fixed in 10% acetic acid and 45% methanol for 30 minutes, incubated in Amplify (Amersham) for 20 minutes, dried, and exposed to Hyper-film (Amersham) for 41 hours at -80°C. (B) COS-7 cells were transfected with pcDNA3.1A C-terminal Myc-tagged ARL5(WT) or ARL5(G2A) and metabolically labeled with [³H]myristic acid before immunoprecipitation of cell extracts with anti-Myc antibody. Beads were dispersed in 20 µl of 2x SDS sample buffer and extracted proteins analyzed by SDS-PAGE in 12% gel. Gels were fixed, dried and exposed to Hyperfilm for 21 days at -80°C (upper panel). Part of each immunoprecipitated sample was subjected to western blotting analysis using anti-Myc (middle panel) and anti-ARL5N antibodies (lower panels). Asterisks indicate the lower bands of both ARL5(WT) and ARL5(G2A).