Figure 2.

Pulse-chase experiments of different recombinant alternative reading frame/core+1 proteins (H77 sequence was used for this study). A: Authentic F[alternative reading frame (ARF)/core+1/F] protein (the empty rectangle marks the sequence overlapping with the core protein while the filled rectangle for the core+1 coding sequence) with a V5 tag (represented by a gray circle) at its C-terminus; B: The ARF/core+1 protein translated from AUG of amino acid 26 with a V5 tag at its C-terminus; C: The ARF/core+1 protein translated from AUG of amino acids 86/88 with a V5 tag at its C-terminus. HuH7 cells were mock-transfected or transfected with various constructs expressing different recombinant ARF/core+1 proteins as indicated. Forty-eight hours after transfection, cells were incubated in methionine-free medium for two hours and subsequently radiolabeled with ³⁵S-methionine in the same medium (160 mCi/mL) for two hours. Then, regular medium with or without MG132 treatment was used for further cultivation. At the indicated times, cells were disrupted and proteins were extracted to perform the immunoprecipitation assay using rabbit anti-F polyclonal antibody.