Figure 3. Quantitative proteomic analysis of pY proteins in the early and the active stages of migratory cells.

(a) Schematic representation of the strategy for the proteomic analysis of the pY proteins. (b) The Venn diagram shows the comparison of the pY proteins identified in the current study with the adhesion related proteins identified by Schiller H. et al.¹², The numbers of uniquely-identified and overlapping proteins from both studies are shown in the diagram. (c) The 2-D scatter-plot shows the spectral counts (x-axis) and MS intensities (y-axis) for all proteins containing quantitative information for all three replicates in at least one time point. The blue dots represent proteins containing quantitative information for all three replicates at only one time point, the red dots represent proteins with quantitative information for all three replicates at both time points. (d) Evaluation of the reproducibility of the quantitation using clustering analysis. The Z-scores of the normalized MS intensities of the 447 proteins with sufficient quantitative information (as indicated by the red dots in panel c) are color-coded to show the relative protein abundance (red: high, blue: low), and hierarchical clustering analysis was performed using the software Perseus with default parameters. Student’s t-test was used to filter out the proteins quantitated with low reproducibility (p < 0.05), which reduced the number of proteins from 447 to 371. The scale bar indicates the Z-scored values of MS intensities. (e) Validation of downregulation of pY-Vimentin at 60 min by immunoprecipitation and Western-blotting. The cells expressing GFP-Vimentin were allowed to adhere on 5 μg/ml fibronectin for the indicated times or kept in suspension (Sus) as indicated, the cells were then harvested and lysed. The fusion protein was immunoprecipitated from the WCLs using anti-GFP antibody, the pY level of immunoprecipitated GFP-Vimentin was detected by Western-blotting using anti-pY antibody, and the total level of GFP-Vimentin was detected by Western-blotting using anti-GFP antibody.