O-linked glycosylation of CD107a is necessary for reduced killing of surface CD107a-expressing cells. sCD107a (triangle) or control (squares) expressing 721.221 cells were incubated for 48 hours in the presence of DMSO (A) or BADG (B), or for 2 hours with NA (C). Left panels: Cells were used in a 4-hour 51Cr release assay with IL-2–activated human NK cells at different E/T ratios. Values represent triplicates ± SD. Right panels: Cells were lysed and analyzed by 2-dimensional anti-CD107a Western blotting analysis. Dotted lines indicate localization of CD107a in DMSO-treated control samples; n = 3. (D) IL-2–activated human NK cells were incubated with HeLa cells stably expressing the indicated sCD107a mutants. Specific lysis was assessed by 4-hour 51Cr release assay. Percentage of inhibition was calculated as stated in Materials and methods. Shown is the mean ± SEM. Each dot represents a single value of triplicates from 4 to 13 experiments of an E/T of 10/1. Significances were analyzed using 1-way ANOVA (P < .0001) followed by Bonferroni’s multiple comparison; ***P < .001. (E) Statistics for Pfn binding ± SEM as analyzed in Figure 3C. Statistical analysis was performed by 1-way ANOVA; n = 8. ns, not significant.
