siRNA target sequences in nef. (A) Targets of siRNAs against the U3-overlapping region of HIV-1_NL432 Nef and their sequences. Nef-expressing HeLa CD4 cells were transfected either with 2.5 μM egfp siRNAs (control: EGFPst) or nef siRNAs (Nef355st or Nef366st). At 48 hours after transfection, these cells were lysed to obtain total RNA and protein. (B) Total RNA was extracted and analyzed by qRT-PCR. The level of nef mRNA expression was normalized with that of elongation factor 1α (EF-1α) mRNA expression (nef /EF-1α). The data represent the expression level of nef mRNA relative to that of the control as 100%. The data represent the average ± SD of 3 independent experiments. (C) The cell lysates were subjected to 12.5% SDS-PAGE and immunoblotted with anti-Nef mAb.