The β₂-m–assembled and free MHC class I HC levels are altered in C282Y mutant cells in an allele-dependent manner. (A) PBMCs with (C282Y) or without (control) the C282Y mutation were ³⁵S-labeled for 2.5 hours and class I molecules recovered with HC-10 and W6/32 mAbs and subjected to 10% SDS-PAGE. The position of the MHC class I HC and β₂-m are indicated. Molecular weight markers are shown on the left. Data shown represent one individual experiment using PBMCs from one WT blood donor and one C282Y homozygous patient with HH of a total of 40 studied. (B) MHC class I HC bands were quantified by phosphoimaging. In each individual experiment one control blood donor and one patient with HH carrying the C282Y mutation were tested. The intensity of the control bands was divided by the C282Y bands. The ratios were plotted according to the patient's HFE genotype: C282Y homozygous (+/+; •/▾); C282Y heterozygous (+/-; ○/▿). ^*P < .05. The means ± SD of 40 experiments with the following distribution are shown: HC-10 (C282Y^+/+), 31; HC-10 (C282Y^+/-), 9; W6/32 (C282Y^+/+), 23; W6/32 (C282Y^+/-), 6. (C) As a control experiment, HLA-DR was recovered from control and C282Y ³⁵S-labeled PBMCs and resolved on a 10% SDS-PAGE. (D) The control/C282Y ratios of the band intensity were plotted according to patient's HFE genotype. Data are representative of 6 individual experiments.