To understand peptide selection by MHC class I molecules on a molecular level, the folding, assembly, and peptide binding of class I molecules have been intensely investigated in recent years. In contrast, the placement of these events into the cellular architecture of the early secretory pathway and their timing in wild type cells are not sufficiently understood, especially with respect to the quality control steps that decide whether class I molecules should be returned to the ER, if bound to low-affinity peptides, or moved to the plasma membrane. In this review article, we focus on a long-established technique to investigate MHC class I cell surface transport: the radioactive pulse-chase assay. We describe the design of experiments and the evaluation of the data, and we point out its potential for future exploration of class I, as well as other methods that can complement and extend it.
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