FIGURE 2.
Spacing of the lysine reside influences MARCH8-induced CD8-DRβ internalization. CD8-DRβ reporter constructs were transfected into 293T cells together with either GFP-MARCH8 or GFP-MARCH8mut. After 24 h cells were harvested and processed for FACS analysis or immunoprecipitation. A, positioning of the lysine residue within the TM and cytoplasmic regions of the various CD8-DRβ reporter constructs. Single letter amino acid code is used. B, summary of surface down-regulation of CD8-DRβ reporter constructs in the presence of MARCH8. Surface CD8 was measured by FACS using anti-CD8 mAb OKT8. The percentage down-regulation was calculated as MFI in the presence of MARCH8wt × 100/MFI in the presence of MARCH8mut. Statistical significance was calculated using a paired t test. Constructs CD8-DRβ-K3 to -K7 were either not statistically different from, or showed only a very small change from CD8-DRβ-wt. Different symbols represent independent data sets. C, FACS analysis of surface CD8 down-regulation using anti-CD8 mAb OKT8. GFP expression identifies GFP/MARCH8- or GFP/MARCH8mut-expressing cells. Histograms show surface expression in the presence of GFP/MARCH8 (hatched) and GFP/MARCH8mut (open). D, immunoprecipitation of CD8-reporter constructs in the presence of GFP/MARCH8 (upper) or GFP/MARCH8mut (lower). Blots were probed with anti-ubiquitin antibody P4D1-HRP. The position of a clear ubiquitin ladder is shown.