Nef isolates 2410 and 2391 both downregulate cell surface CD4 but differentially downregulate cell surface SERINC5. CD4+ HeLa cells expressing 2410 or 2391 Nef-eGFP and SERINC5.intHA were stained for cell surface CD4 and SERINC5 and analyzed by flow cytometry. CD4+ HeLa cell populations were determined by gating on eGFP+ cells using an FMO control. (A and B) Representative pseudocolor plots illustrating cell surface SERINC5 (Alexa Fluor 647) (A) and cell surface CD4 (PE) (B) levels of Nef+ cell populations (eGFP+). (C and D) Representative histograms illustrating cell surface SERINC5 (C) or CD4 (D) levels on CD4+ HeLa cells after gating on single and Nef+ (eGFP+) cells. Geometric mean fluorescence intensities (MFIs) of the cell surface proteins are indicated. (E to G) Summary of the fold downregulation ability (±SE) for cell surface SERINC5 (E), cell surface CD4 (F), and fold Nef-eGFP (G) expression (n = 3). (H) Western blot illustrating the expression of Nef-eGFP fusion proteins in the absence and presence of SERINC5 in CD4+ HeLa cells. eGFP, enhanced green fluorescent protein; AF647, Alexa Fluor 647; PE, phycoerythrin; SE, standard error; Mw, molecular weight; kDa, kilodaltons; NT, nontransfected; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ***, P ≤ 0.001; ****, P ≤ 0.0001; n.s., nonsignificant (P > 0.05).