Figure 4.

ASIC1a is expressed in mitochondria of mouse cortical neurons. (a) Presence of ASIC1a and organelle markers (PDI for ER, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for cytosol, and VDAC and Cyt C for mitochondria) in cell fractions of mouse cortex separated by centrifugations at different speeds ( × 1000 × g) as indicated. (b) Fluorescence images of a cultured mouse cortical neuron co-transfected with EGFP-ASIC1a (green) and Mito-DsRed (red). Insets, enlarged images for the boxed region. Scale bar=10 μm. See Supplementary Figure 4 for details of colocalization. (c) Presence of ASIC1a and organelle markers in mitochondrial preparations by Percoll gradient centrifugation. Total, whole cortex lysate; P1, crude nuclear fractions; Mito, mitochondrial fractions; P2, light membrane fractions; Cyto, cytosolic fractions. (d) Presence of ASIC1a and VDAC in total lysates (total) and mitochondrial fractions (Mito) prepared from brains of ASIC1a^+/+ and ASIC1a^−/− mice. (e) Presence of ASIC1a and ANT1/2 in mitoplast fractions (CM, crude mitochondrial fractions; PM, purified mitochondrial fractions; MP, mitoplast fractions). (f) Fluorescence images of crude mitochondria isolated from CHO cells expressing EGFP-ASIC1a (green) and Mito-DsRed (red). Scale bar=5 μm. (g and h) Detection of GFP signals in total, mitochondrial (Mito) and cytosolic (Cyto) fractions of CHO cells that were separately transfected with GFP-ASIC1a or EGFP-vector (g) and ΔN-EGFP-ASIC1a or ΔC-EGFP-ASIC1a (h). Cytochrome c oxidase subunit 2 (MTCO2) was used as a marker for mitochondria