Association of βPIX with KIF5A in the rat brain. (A) Mass spectrometry. Anti-βPIX immunoprecipitates were silver stained (left). Marked silver-stained bands (arrowheads) were subjected to mass spectrometry followed by immunoblotting (right, three columns). (B) Immunoblotting for the indicated proteins following immunoprecipitation with anti-βPIX, -KIF5A, or -KLC antibody. (C) GST pulldown assay. His-tagged proteins (input and bound), Glutathione-Sepharose immobilized GST-βPIX or GST protein (bead) were immunoblotted by anti-His, or GST antibody. (D) Representative FRET images from primary cultured rat hippocampal neurons co-transfected with plasmids encoding the indicated pairs of proteins. Acceptor (YFP)-bleached areas were indicated by white boxes. Negative control, CFP/YFP and βPIX-CFP/KIF3A-YFP; positive control, GIT1-CFP/βPIX-YFP. (E) Quantification of FRET efficiencies (mean ± SEM.). N = 20 cells/each condition. Student’s t-test. *P = 0.0124, ***P < 0.0001.