EAAC1 and NCX1 are assembled into a multimolecular complex in mitochondria.

<p>(A) Selective NCX1 immunoprecipitation by EAAC1 antibody in mitochondrial protein extracts from rat cortex and hippocampus. Three different western blots experiments using NCX1, NCX2 or NCX3 antibodies are reported. The gels were loaded with mitochondrial crude protein extracts (mt) or with EAAC1-immunoprecipitated proteins (mt-IP EAAC1). For western blots resolved with NCX1 antibody, brain tissue homogenate (brain) and normal mouse serum-immunoprecipitated proteins (mt-IP NM) were used as a positive and a negative control, respectively. For NCX2 and NCX3, proteins from isolated membranes (m) or EAAC1-immunoprecipitated membranes (m-IP EAAC1) were also analyzed. (B) Mitochondrial protein extracts (from the same preparations used in panel A) immunoprecipitated with NCX1 antibody and resolved with GLAST, GLT1 and EAAC1 antibodies. Brain tissue homogenate (brain) and normal mouse serum-immunoprecipitated proteins (mit-IP NM) were used as a positive and a negative control, respectively. (C) Immunoprecipitations with EAAC1 and NCX1 antibodies in SH-SY5Y and C6 cells, respectively. In the immunoblot analysis with NCX1 antibody the IP lane corresponds to EAAC1-immunoprecipitated protein extracts; in the EAAC1 immunoblots the IP lane corresponds to NCX1-immunoprecipitated protein extracts. Protein extracts from rat brain tissue homogenate and from BHK cells stably expressing NCX1 were used as controls. In each IP lane, the lower band at around 50 KDa represents the immunoglobulin. (D) Results from confocal microscopy analysis using NCX1 antibody and the mitochondrial marker MitoTracker on rat hippocampal and cortical mitochondria are shown on the left. Results on the same mitochondrial preparations probed with EAAC1 (red) and NCX1 (green) antibodies are shown on the right. Note the strong overlap of the two signals in the merged image.