Phosphorylation of the Ser residue on IRS1 changed the topog

Phosphorylation of the Ser residue on IRS-1 changed the topography of IRS-1, causing it to be degraded by a different signaling pathway. In eukaryotes, the protein machine that degrades proteins is called the proteasome. Given the fact that pharmacological molecules that are capable of inhibiting the proteasome are available to researchers, design a simple experiment to prove your hypothesis.   Be sure to show predicted results. (Answrs should be what data should look like)

Solution

Measure the fraction of newly synthesized proteins which are degraded by proteasomes. The strategy is to assay newly synthesized proteins in the presence and in the absence of inhibitors of proteasome function. Pulse-label mouse lymph node cells with methionine (35S) and then chase with excess unlabeled methionine for 30 minutes, all in the presence or absence of proteasome inhibitors. At different times during the chase, take aliquots of cells and boil them in SDS solution to denature all proteins. For each sample, load identical amounts of cellular protein and run onto SDS-PAGE followed by quantification of the radioactivity in each lane.

Predicted results: In this experiment the plateau value for the radioactive proteins in the absence of proteasome inhibitors was slight lower than it was in the presence of inhibitors, indicating that the newly synthesized proteins are degraded in proteasomes in lymph node cells.