Fig. 2.

Distribution of PFAS, PAICS and ATIC in cultured rat hippocampal neurons. A Representative example of a hippocampal neuron co-labeled with PFAS (green) and a mitochondrial marker Tom20 (red). Scale bar, 10 μm. B Enlarged view of the boxed region (dendrite) in A illustrating close proximity or co-localization between PFAS (green) and mitochondria (red). Image shown is a single middle section of z-scans of the dendrite. Sample fluorescence intensity profiles along dashed lines are shown on the right. C Example of a segment of a dendrite co-labeled with PFAS (green) and another mitochondrial marker, CoxIV (red). D, E Examples of dendrites from neurons co-labeled with PAICS (green) and either Tom20 (red) or CoxIV (red). F, G Examples of dendrites co-labeled with ATIC (green) and either Tom20 (red) or CoxIV (red). Fluorescence intensity profiles show various extents of overlaps between green PFAS, PAICS or ATIC and red mitochondria. Scale bar in G is 1 μm and applies to B–G See also Supplemental figure 1. H–J Quantification of PFAS, PAICS or ATIC puncta in close contact (pixel touching to<50 pixel overlapping) or co-localized (≥50 pixel overlapping) with Tom20- or CoxIV-labeled mitochondria. K Immunoblots showing the presence of PFAS, PAICS and ATIC in mitochondria isolated from cultured hippocampal neurons. Fifteen micrograms of proteins from neuronal lysates (total), isolated mitochondria (mito) and mitochondria-depleted cytosol (cyto) were analyzed by immunoblot with the indicated antibodies. Note that the purity of isolated mitochondria is illustrated by the enrichment of a mitochondrial marker VDAC, and the absence of cytosolic marker HSP90 and MEK1/2 as well as nuclear marker hnRNP. See also Supplemental figure 2 (Color figure online)