HLA-B*57:01 binds to abacavir during intracellular maturation. Panel a shows flow cytometric evaluation of HLA expression on the cell surface. HeLa cells, in which transfection of HLA-B*57:01 or B*57:03 and exposure to brefeldin-A were performed simultaneously, were subjected to flow cytometry for HLA assessment. Panels b, c, and d present LC-MS/MS chromatograms of standard and immunoprecipitated abacavir samples b), concentrations of abacavir and FLAG-tagged HLA expression in FLAG-immunoprecipitated samples c), and abacavir concentration per unit of immunoprecipitated FLAG-tagged HLA in FLAG-immunoprecipitated samples d). Lysates (containing 1.5 mg of protein) from HeLa cells, in which transfection of HLA-B*57:01 or B*57:03 and exposure to brefeldin-A as well as 100 μM of abacavir were performed simultaneously, were subjected to immunoprecipitation of FLAG-tagged HLA. A total of 120 μL of immunoprecipitated samples were obtained. FLAG-tagged HLA expression in FLAG-immunoprecipitated samples was assessed via immunoblotting and analyzers. N.D. indicates “not detected” c) and “not determined” d).