Panel A: Experimental schematic shows skin fibroblasts treated with I F N beta and I F N gamma followed by R T-q P C R, western blot, and activity assays. Panel B: Western blot and vertical bar graphs show reduced expression of P S M B 8, P S M B 9, and P S M B 10 in mutant cells after interferon stimulation. Panel C: Activity-based probe assays and vertical bar graphs show decreased proteasome activity in P S M B 10 G 209 R compared to wild-type cells.
Reduced immunoproteasome activity in IFN-stimulated PSMB10 G209R skin fibroblasts. (A) Primary neonatal skin fibroblasts isolated from the patient with the PSMB10 G209R mutation and age-matched neonatal primary PSMB10 WT skin fibroblasts were treated with 75 U/ml IFNγ or 100 U/ml IFNβ using cells from passages 4–10 for RNA, protein, and activity analyses (scheme was prepared using BioRender). (B) Western blot analysis of immunoproteasome subunits PSMB8-10 with β-actin as a loading control. The two bands for PSMB10 represent the pro- and the mature forms. Upon IFN stimulation, expression and integration of PSMB10 into an active 20S proteasome are enhanced as evidenced by the presence of mainly mature PSMB10, which is generated upon assembly into the 20S and proteolytic removal of its propeptide. Representative western blot is shown, and densitometric analysis of 3–4 independent experiments where relative expression of PSMB8, PSMB9, and PSMB10 to β-actin was normalized to the PSMB10 WT control (multiple unpaired t test, P value: *** <0.001, **** <0.0001). (C) ABP analysis of proteasome activity using the pan-reactive probe MV151, PSMB6- and PSMB9-specific probe LW124, and PSMB5- and PSMB8-specific probe MVB127 (n = 6–7, multiple unpaired t test, P value: * <0.05, *** <0.001, **** <0.0001). The β-actin loading controls shown in B and C and Fig. S1 B are identical, as these panels were derived from the same experiment and immunoblot. Source data are available for this figure: SourceData F4.