The R5 AIDS Nef from HIV-1 clone *E11 does not down regulate CD4 more than the two pre-AIDS alleles from 32D2 and 8G9, but does downregulate MHC Class I more than the pre-AIDS Nefs. SupT1 cells were electroporated with 10 μg of pA-Nef expression vectors and 2 μg of pCMV-EGFP expression vector. Cells were analyzed by flow cytometry 24-hrs post-electroporation. The fraction of control levels of cell surface CD4 (A and C) or MHC Class I A2 (B and D) expression in GFP+ cells are reported for each allele. C and D 2.5, 10 or 20 μg of pA-*E11 Nef (diamonds), pA-32D2 Nef (squares), or pA-8G9 Nef (triangles) were transferred to SupT1 cells by electroporation. The average of eight transfections for A and B or two transfections for C and D is shown. Error bars represent the standard errors of the mean. Samples denoted with asterisks were significantly different from the *E11 sample as determined by the Student's unpaired t-test (A and B) or by the Student's paired t-test (C and D). E Twenty μg of pA-Nef expression vectors were used for electroporation of SupT1 cells with 2 μg of pCMV-EGFP. Cells were lysed in sample buffer and analyzed by SDS-PAGE and western blot. The blot was probed with a polyclonal rabbit anti-Nef serum followed by 125I-Protein A. The blot was then analyzed by phosphorimager and quantitated using ImageQuant software. Results from a representative experiment of three experiments performed are shown.