Figure 4

In situ PS4≡ protein profiling during HCV replication identifies nine differentially active proteins. The PS4≡ probe was added to the cellular media of naïve Huh-7 cells (A) or Huh-7 stably expressing the HCV replicon (B) for 1 h at 37°C. The labeled proteome was isolated and subjected to Huisgen's 1,3-dipolar cycloaddition with rhodamine azide. After separation of the proteomes by two-dimensional gel electrophoresis, the fluorescently tagged proteins were scanned (A, B), silver stained (C, D), and identified by LC-MS/MS (Table 1). (E) The differential activity between in situ (Figure 4) and in vitro (Figure 3) fluorescently labeled proteins in Huh-7 cells was quantified with the ImageJ software (NIH) by measuring the fluorescence intensity (normalized for volume) of the corresponding protein spot. (F) The differential activity of fluorescently labeled proteins between HCV and naïve Huh-7 cells was quantified with the ImageJ software (NIH) by measuring the fluorescence intensity (normalized for volume) of the corresponding protein spot of HCV replicating Huh-7 cells (B) versus of that of naïve Huh-7 cells (A). Statistically significant differences were determined using ANOVA, with probabilities *p < 0.05.