Fig. 6.

Deletion of cytoplasmic lysines leads to increased surface abundance of ISG75. (A) Localization of ISG75L, ISG75dK12, ISG75dK345, and ISG75dK in nonpermeabilized cells were visualized using mouse anti-HA antibody and fluorescein isothiocyanate-conjugated secondary antibody (green). Gated, reduced exposure time to obtain a more resolved image. Cell morphology is shown by phase contrast. DNA was detected by counterstaining with DAPI (blue). Bar, 2 μm. (B) Cells were further imaged under permeabilized conditions using confocal microscopy. Bar, 7.5 μm. (C) Surface biotinylation was used to determine the ratio of proteins on the surface to proteins in the intracellular pool. Wild-type ISG75L reporter was found mostly in the intracellular pool, with only ∼20% was found on the surface, whereas the ISG75dK lysine-null mutant saw an increase in surface representation (∼50%). The graph shows the percent biotinylation of ISG75 reporters and represents the means of three completely independent experiments, with the standard deviations indicated.