Recognition of endogenously derived epitope. (A) Vector construction. The vector expresses EGFP as a transfection marker and a fusion protein that consists of mini-Nef gene coding 31 amino acids of Nef123-Nef153 region and GlyGlyGlyGlySer linker and renilla luciferase. The 133I/135F and 133T/135F mutant vectors as well as wild type vector were constructed. Mini-Nef sequence that corresponds to the actual Nef gene is shown. (B) By measuring luciferase activity, quantity of the generated mini-Nef was assessed. No significant differences in mini-Nef generation between wild type, 135F and 133T/135F were observed. Each stick and bar indicates average and standard deviation of three independent experiments. (C) The vector transfected cells were incubated with either H27-9 or I30-1, and IFN-γ secretion was quantified by ELISA and normalized to determine the epitope recognition. For normalization, each value was divided by IFN-γ secretion with wild type peptide concentration of 9 μM. Mock as well as mGag, transfected with mGag(wt)-hRluc-EGFP, were measured as negative controls. Each stick and bar indicates average and standard deviation of three independent experiments. The significance was calculated by unpaired Student’s t-test; **, p < 0.01; ***, p < 0.001.