Figure 3
Individual gel analysis of affinity tagged PINCH protein complexes. Separate pull-down samples were analyzed in individual 2D gels, and visualized using Deep Purple fluorescent stain: Flag-PINCH (A), PINCH-PrA (B), and Flag control (C). Pull-down experiments were repeated in triplicate, and compared in a Venn diagram (D). Nine spots appeared reproducibly regardless of the tag employed, and were absent in control gels. E, A representative Flag-PINCH gel showing nine PINCH-specific spots of interest (circled in white). The numbers are derived from the master catalog used to match coordinates of all gels. Spot 54 and 113 were identified as ILK and RSU-1 by 2D immunoblot, respectively (see Additional file 5: Figure S2). The boxed area is highlighted in panel F. F, Independent pull-down experiments were performed comparing PINCH to PINCHQ38A, which disrupts the PINCH-ILK interaction. MS analysis revealed that spot 64 contained Parvin, and spot 64 is absent in the PINCHQ38A purification, as indicated by the arrow.