Figure 3

H-rev107 protein inhibited RAS palmitoylation. (A) Coomassie blue staining of purified full-length and C-terminal-truncated H-rev107 from E. coli. The 6 × His-tagged fusion proteins of H-rev107 were purified using a HiTrap™ Chelating HP column. The purified proteins were subjected to 12% SDS-PAGE. (B) HtTA cells plated in a 6-cm dish were transfected for 24 h with 7 μg of H-rev107 (FL) or R-phycoerythrin or 3 μg of H-rev107 (1–132) protein. Cell lysates were prepared, and acyl-biotin exchange analysis of H-RAS was performed as described in Methods. Acylated RAS from 5 μg of cytosolic extract was collected with streptavidin agarose resin followed by analysis by Western blotting. The loading input consisted of 300 ng of protein from the acyl-biotin exchange. FL: full length.