Figure 7
Validation of TGF-β induced changes in protein expression levels identified by WGA affinity workflow. (A) The ratio of peptide abundance identified by selectively monitoring three transitions from a peptide by multiple-reaction monitoring (MRM, shown as black bars) derived from each of six proteins is compared to the results from the original analysis of the first three biological repetitions (BR1, white bars; BR2, gray bars, and BR3, hashed bars). Protein ratios are shown as log2 of the ratio of abundance in TGF-β treated NMuMG cells relative to non-treated CTL cells. MRM results are as follows when presented as fold-change ± standard deviation, similar to those shown in Table 2: Neural cell adhesion molecule 1 (NCAM; 2.82 ± 0.22), Fibronectin (3.74 ± 0.51), Fibulin-2 (1.86 ± 0.10), Pantetheinase (0.38 ± 0.03), Galectin-9 (0.33 ± 0.10), and α-N acetylglucosaminidase (0.46 ± 0.04). (B) Western blot analysis of unfractionated cell lysate (IN), WGA flow-through (FT) and elution (EL) fractions of JM01 and NMuMG cells cultured in the absence (-) or presence (+) of TGF-β1 for 24 hrs. The protein levels of Integrin β4 (JM01), Basigin, and Integrin α3 all reduced upon treatment of the cells with TGF-β1, whereas those of Integrin β5 (JM01) increased. Using immunofluorescence microscopy (C, D) the occurrence of EMT in the JM01 and NMuMG was confirmed by the reorganization of the F-actin filaments (red, top panels), and is accompanied by the TGF-β induced upregulation of Clusterin and down-regulation of Integrin α6 in the JM01 cell line (C), and the up-regulation of Fibronectin and NCAM in the NMuMG cell line (D).