sdAb19 inhibits Nef-induced CD4 down-regulation. HPB-ALL cells were transfected with plasmids for the expression of either Nef-GFP or GFP in combination with increasing amounts of the plasmid for expression of sdAb19. (A) sdAb19 activity on Nef-induced CD4 cell surface down-regulation. Transfected cells were stained with PE-Cy5–conjugated anti-CD4 at 4°C, and surface expression of CD4 in Nef-GFP– or GFP-expressing cells was measured by flow cytometry. Results are expressed as the percentage of the MFI determined in GFP-positive cells relative to that determined in GFP-negative cells. Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. The NefLL/AA mutant (▩) mutated in the AP-binding di-Leu motif (LL164/165) was used as negative control. (B) sdAb19 activity on Nef-induced acceleration of CD4 internalization. Transfected cells were stained at 4°C with PE-Cy5–conjugated anti-CD4 and then incubated at 37°C for 5 or 15 minutes to allow internalization. Cell surface-bound anti-CD4 was then stripped by acidic wash, and cell-associated CD4 staining was measured by flow cytometry on GFP-positive cells. The percentage of internalized CD4 at each time point was calculated as described.⁴³