Fig 2: Identification of the differentially expressed proteins by the iTRAQ-based strategy. (A) Schematic diagram of the workflow for the iTRAQ-based experiments. Six dishes of HepG2 cells were pooled into one sample for either normal (Ctrl) or Leu-deprived (-Leu) HepG2 cells. Then pooled cells were fractionated, digested into peptides, labeled with different iTRAQ reagents containing reporter groups of different masses (113, 114), balance groups of different masses (192, 191), and a reactive group (R). The labeled peptides were then mixed equivalently, fractionated by strong cation exchange (SCX), and analyzed by LC-MS/MS. Two independent biological replicates were performed for the iTRAQ experiments. (B) The protein distribution in accordance to Log10 theoretical weight (MW; Da) versus log2 Ratio of all the differentially expressed proteins in -Leu/Ctrl. Red dots indicate up-regulated proteins, green dots indicate down-regulated. (C) Validation of the differential expressions of ADH1C and ALDH1A1 by immunoblotting. (D,E) Quantitation of ADH1C/ß-Actin and ALDH1A1/ß-Actin as described in (C). Data are means ± SD (n = 3). * P < 0.05, ** P < 0.01 (paired Student’s t-test).