Figure 3.

Subjects with BAP1 variants exhibit increased amounts of Ub-H2A and pro-β5 proteasome subunit

(A) Top: whole-cell lysates from T cells of affected individuals carrying the p.Pro12Thr or p.Cys91Ser BAP1 variants (labeled probands 1 and 5) and control T cells (subjects’ father and/or mother) were subjected to protein extraction and SDS-PAGE/immunoblotting with antibodies directed against Ub-H2A, H2A as well as α-tubulin and GAPDH (loading control), as indicated. Bottom: densitometry analysis of the shown immunoblots (top) depicting the Ub-H2A contents detected in control (black) and BAP1 subjects (red). The y axis represents the fold changes of normalized Ub-H2A (Ub-H2A/α-tubulin) in densitometry measurements, which were set as 1 for subjects’ father or mother, as indicated.

(B) Native-PAGE analysis from control and affected individuals’ T cells with proteasome bands being visualized by exposing the gel to 0.1 mM of the LLVY fluorescent peptide (left) and staining the gels with a monoclonal antibody specific for the α6 subunit (right), as indicated.

(C) Top: whole-cell lysates from control and affected individuals’ T cells were separated by SDS-PAGE and analyzed by immunoblotting with antibodies directed against, RPT1, RPT2, RPT3, RPT4, RPT5, RPT5, β1, β2, β5, and β5i, as indicated. Equal loading was ensured by probing the membranes with an anti-GAPDH antibody, as indicated. Bottom: densitometry analysis of the shown immunoblots (top) depicting the pro-β5 contents detected in control (black) and affected individuals (red). The y axis represents the fold changes of normalized pro-β5 (pro-β5/GAPDH) in densitometry measurements, which were set as 1 for subjects’ father or mother, as indicated.