FIGURE 4.

Steady-state ISCU and NFS1 protein levels are inherently low in muscle tissue. Protein levels in lysates from control (Ctl) and patient (Pat) vastus lateralis biopsies and primary cells were assessed by Western blots. A, Fe-S assembly proteins ISCU and ISCS. Short and long film exposures are shown for completeness. B, ISCU mRNA levels were assessed by Northern blot in a healthy control vastus lateralis biopsy and in primary cells derived from a healthy individual. Methylene blue-stained 18 S and 28 S ribosomal RNA bands served as loading control. C, Western blots assessed levels of Fe-S assembly cofactors ISD11 (LYRM4), Fxn, and NFS1 in control and patient muscle biopsies. D, Fe-S recipient proteins succinate dehydrogenase subunit B and mitochondrial aconitase (mACO). All proteins above were probed on the same filter, and citrate synthase served as a loading control for mitochondrial protein. E, expression of the Fe-S assembly proteins ISCU and NFS1, as well as the Fe-S recipient proteins succinate dehydrogenase subunit B and mitochondrial aconitase in multiple mouse tissues was evaluated by Western blotting. F, siRNA-mediated knockdown of ISCU protein in control patient myoblasts led to decreased NFS1 and ISD11 protein levels, whereas FXN levels increased. Citrate synthase and mitochondrial complex IV, subunit IV (CIV-IV) proteins served as loading controls.