Determining the Half-Life of MHC Class I Molecules by Blocking Protein Synthesis Using Cycloheximide

Major histocompatibility complex class I (MHC I) molecules play a crucial role in activating adaptive immune responses by presenting viral and tumor antigens to cytotoxic CD8⁺ T cells. Additionally, MHC I molecules have been implicated in autoimmunity through the presentation of self-peptides. The effective presentation of antigenic peptides depends on the stability of MHC I allotypes. Evaluating MHC I stability involves analyzing its degradation kinetics and determining its half-life. In this chapter, we present an assay to evaluate the degradation kinetics of MHC I molecules (both endogenous and overexpressed) in cell lines by inhibiting protein synthesis with cycloheximide. Additionally, we incorporated MG132, a proteasomal degradation inhibitor, to examine the impact of the proteasome on MHC I degradation. Furthermore, we detail a method for calculating the half-life of MHC I molecules by fitting the degradation data into a one-phase decay model.