Figure 2.

Gas7 detection and localization with mitochondria. A, Sucrose gradient fractionation of mouse cerebrum homogenate. Homogenized solutions were subjected to ultracentrifugation in 15%‐35% sucrose gradients. Fractions were then resolved by SDS‐PAGE and immunoblotted with an antibody against Gas7. Gas7 was detected in fractions 2‐4 and 14‐17, SDHA in fractions 3 and 4 and 14‐17, GM‐130 in fractions 2 and 3, and actin and Gapdh in fractions 14‐17. B, Immunoblots of Gas7, SDHA, and GAPDH of isolated mitochondrial, cytosolic, and total lysate fractions from WT cerebrum showing that Gas7 is distributed in cytosol and mitochondrial fractions of WT mice. SDHA and GAPDH were used as markers for mitochondrial and cytosolic fractions, respectively. C, Quantification analysis of Gas7 showing that Gas7 is detected in both cytosol and mitochondria fractions. Gas7 detected in the abovementioned fractions was normalized with markers for mitochondrial (SDHA) and cytosolic (GAPDH) fractions, respectively. D, Immunofluorescence analysis of Gas7 and mitochondria colocalization in primary cortical neurons. Top panel shows Gas7 (red) labeled with Gas7 polyclonal antibodies and mitochondria (green) are labeled with SDHA antibody. Bottom panel shows the colocalization (yellow) of Gas7 and mitochondria in a merged image