Fig. 2

Enhanced ATAD3A oligomerization in HD models. a Western blot (WB) analysis of rat liver mitochondrial fractionations with the indicated mitochondrial antibodies. OM: mitochondrial outer membrane. CS: contact sites. IM: mitochondrial inner membrane. b HdhQ7 and HdhQ111 cells were stained with anti-ATAD3A and anti-Mitofilin (a mitochondrial contact site protein) or anti-Cyto C (a mitochondrial intermembrane space protein) antibodies. Nuclei were stained with DAPI. In situ Duolink proximity ligation assay (PLA) was performed. Histogram: quantitation of the number of PLA-positive signals (red). At least 200 cells/group were analyzed. 3 independent experiments, unpaired Student t-test. Scale bar: 10 µm. c Immunogold electron microscopy analysis with an antibody directed against ATAD3A N-terminus was performed in HdhQ7 and HdhQ111 cells. Bottom images are the images boxed. Scale bar: 100 nm. Blue dash line: OMM, mitochondrial outer membrane. Yellow dash line: IMM, mitochondrial inner membrane. Red arrowhead: ATAD3A. Histogram: the percentage of ATAD3A immunopositive gold particles on the contact site versus total number of gold particles. 3 independent experiments, unpaired Student t-test. d Left: ATAD3A protein level was determined by WB in the presence or absence of β-ME. Right: Protein lysates were incubated with the crosslinker BMH (1 mM). e HdhQ7 and Q111 cells were treated with BMH (1 mM, 20 min). f Total lysates of striatum from YAC128 (12 months old), R6/2 (12 weeks old), or age-matched wildtype mice were analyzed by WB with anti-ATAD3A antibody in the presence or absence of β-ME. n = 6–10 mice/group. g ATAD3A oligomers were analyzed in HD patient fibroblasts (HD1: GM21756, Female; HD2: GM04693, Male; HD3: GM21756, Female) and normal subjects (Con 1: nHDF, fibroblasts from juvenile; Con 2: HDF, fibroblasts from adult; Con 3: Huf1822, adult). h ATAD3A oligomers were analyzed in total lysates of HD patient postmortem brains (cortex: left; caudate nucleus: right) or normal subjects, under non-reducing condition. [Cortex: Normal X5302; HD X5298. Caudate nucleus: Normal 5214; HD Grade1 (G1): 4283; HD Grade2 (G2): 4557; HD Grade3 (G3): 3573]. See Supplementary Fig. 1F for patient information. The data are mean ± SEM. Shown representative blots are from at least 3 independent experiments