FIG 7.

Palmitoylation of F13 occurs in the presence of Retro-2. (A) HeLa cells on coverslips were infected with 3 PFU/cell of VACV F13-GFP and after 1 h incubated with 100 μM palmitic acid-azide and 20 μM Retro-2 or DMSO carrier. At 8 h after infection, the cells were washed, fixed, and labeled by Click-It chemistry with alkyne-Alexa Fluor 594. Cells were examined by confocal microscopy, and superimposed z-stack projections are shown. Colocalizations were determined on individual focal planes using IMARIS software prior to stacking. Green, GFP; red, palmitate; yellow, colocalization. (B) HeLa cells were infected and labeled as for panel A. The cells were lysed, and F13 was captured with antibody to GFP attached to protein G on magnetic beads (lanes 5, 6, and 8) or control IgG (lane 7). After washing, biotin was attached with Click-It chemistry. Proteins were eluted, resolved by SDS-PAGE, blotted onto a membrane, and probed with streptavidin conjugated to IRDye 800. In lane 8, copper was omitted from the Click-It reaction mixture to provide a control.