Figure 3

PS4≡ protein profiling is activity specific in hepatoma Huh-7 cells. The active proteome (200 μg) isolated from naïve Huh-7 cells (B) and Huh-7 cells stably replicating HCV (C) was labeled in vitro for 1 h at 37°C with 20 μM of PS4≡ probe. A representative gel of the denaturing preheating step of 100°C for 5 min (Δ) was performed as a control (A) to confirm the PS4≡ probe specificity toward active enzymes. After separation of the proteomes by two-dimensional gel electrophoresis, the fluorescent-tagged proteins were scanned for fluorescence (A - C) and subjected to silver staining (D - F). The labeled proteins, indicated with arrows, were compared with in situ profiling (Figure 4) and sent for identification by LC-MS/MS (Table 1). The other unmarked fluorescent spots were considered non-specific background as they didn't meet the inclusion criteria of a MASCOT score above 50, a corresponding pI and molecular weight, as well as being identified in at least three independent experiments. (G) The differential activity of fluorescently-labeled proteins in vitro 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 (C) versus of that of naïve Huh-7 cells (B). Statistically significant differences were determined using ANOVA, with probabilities *p < 0.05.