Identifying Protein-Protein Interactions.

<p>(a) Immunoprecipitates of phosphorylated proteins from T-47D (Manassas, VA) lines grown in media, treated with Heregulin for 5 minutes, and 3 hours, and control, in media without Heregulin, are mixed together, run on an SDS-PAGE gel and transferred overnight. The slides are probed with anti-ubiquitin (blue) and anti-phosphotyrosine (red). (b) The experiment is repeated, with cells treated with Heregulin for 3 hours. (c) The images from (a) and (b) are separated by color channel, thresholded, and filtered. (PTyr = anti-Phosphotyrosine, Ub = anti-Ubiquitin). The images are horizontally averaged across the gel lane, and plotted as a function of position. Clear differences between the phosphorylated states can be seen. The PTyr and Ub traces are markedly different, indicating that a distinct subset of phosphorylated proteins are ubiquitinylated. Since each lane is mixed with an equal quantity of control lysate, the differences shown here are solely due to the changes in phosphorylation from 5 minutes to 3 hours of treatment with Heregulin. Additionally, the ubiquitin traces can be seen to clearly overlap selected phosphorylation peaks, indicating proteins that have dual post-translational modifications.