Figure 5. .

Subcellular location of ACAD9 protein. a and b, Western blot of fibroblast and liver lysate with anti-human ACAD9 antibodies. One major band (M) in control samples (a, lanes 2 and 4; b, lane 2) co-migrated with purified recombinant mitochondrial ACAD9 (a, lane 1; b, lane 1), and another was larger (c/n). Both forms were missing or reduced in patient samples (a, lanes 3 and 5; b, lane 3). VLCAD-deficient fibroblasts (V) (a, lane 2) and normal human fibroblast (N) (b, lane 2) or postmortem liver (N) (a, lane 4) served as controls. c–e, Immunofluorescent staining of normal fibroblasts with ACAD9 (c, green) and mitochondrial ATP synthesase (mATPase) (d, red) antibodies, visualized by fluorescent secondary antibodies. The merged image (e) shows that the two proteins colocalize in part (yellow). A green ACAD9 signal is seen in the nucleus (white arrow). Panels f–h and i–k are the same as panels c–e but use fibroblasts from patients 2 and 1 (P2 and P1), respectively. l–n, Immunofluorescent staining of postmortem liver from patient 3 (P3) and a control (C) with antibodies against ACAD9 (l, green) or liver-specific α1-AT (m, red), visualized by fluorescent secondary antibodies. The merged signal is shown in panel n. ACAD9 (αA9) (lanes 1 and 2) staining is deficient in patient 3 (lane 2), but MCAD (αM) (lane 3) and VLCAD (αV) (lane 4) staining are normal. o–t, Immunofluorescent staining of postmortem cerebellar cortex of patient 1 (P1) (o–q) and control (r–t) with antibodies to the cerebellar cortex marker EAAT4 (o and r, red) or ACAD9 (p and s, green); the merged signal is shown in panels q and t, in pseudocolored yellow. ACAD9 is prominent in control (s) but is absent in patient cerebellum (p). A white arrow indicates ACAD9-specific staining of dentate nucleus in deep white matter of the normal control.