PSMB10 p.Ser208Phe variant is associated with decreased immunoproteasome expression, reduced PA28–proteasome complexes, diminished proteasome activity, and disrupted protein homeostasis in PIII-derived T cells. (A) T cells expanded from PBMCs of patient PIII and two age-matched healthy male and female donors (HD) were subjected to protein extraction, followed by native-PAGE separation and in-gel overlay assays (LLVY-AMC) to assess proteasome activity, as well as western blotting using PSMA1- and PSME1-specific antibodies. Lines indicate the migration positions of the 30S, 26S, and 26S–PA28 (hybrid) complexes, as well as the 20S and 20S–PA28 complexes, and the free PA28 heptameric ring. (B) Protein lysates from A were analyzed by SDS-PAGE and western blotting using antibodies directed to K48-linked ubiquitinated proteins PSMB8, PSMB9, PSMB10, PSME1, and GAPDH, with GAPDH serving as a loading control. Shown is one representative experiment (left) and densitometric analyses (right), with data expressed as fold-change mean values (n = 3) relative to a replicate of HD1, which was set to 1 after normalization to GAPDH. Statistical significance was assessed using a paired t test (*P < 0.05 and **P < 0.01). Source data are available for this figure: SourceData F3.