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Fig. 9. An amino-terminal fragment of CAP350 localizes to subnuclear foci and recruits PPAR ${alpha}$ to these foci. NIH3T3 cells were cotransfected with expression plasmids for EYFP-CAP350_1-890 (green) and mRFP-PPAR ${alpha}$ (red) (A-C) or for EYFP-CAP350_1-890(LSHAA) and mRFP-PPAR ${alpha}$ (D-F) and subjected to live cell imaging. Panels C and F show the merged images of panels A and B and panels D and E, respectively. Representative images are shown. EYFP-CAP350_1-890 and EYFP-CAP350_1-890(LSHAA) concentrate in subnuclear foci but are not present in the centrosome or in cytoskeletal microfilaments. mRFP-PPAR ${alpha}$ colocalizes in subnuclear foci with EYFP-CAP350_1-890 but not with EYFP-CAP350_1-890(LSHAA) (panel F). Bar, 10 µm. (G) Mutation of the LXXLL motif in the amino-terminal fragment of CAP350 does not abrogate its binding to PPAR ${alpha}$ in vitro. In vitro synthesized, L-[³⁵S]methionine-labeled PPAR ${alpha}$ or firefly luciferase (Luc) (a control for nonspecific protein binding) synthesized in vitro was incubated with immobilized GST-CAP350_1-890 or GST-CAP350_1-890(LSHAA), as indicated, and bound radiolabeled proteins were analyzed by SDS-PAGE. The left lanes show parallel binding reactions carried out with labeled luciferase as a negative control. Lanes designated Load (1/10) had 10% of the L-[³⁵S]methionine-labeled protein added to each of the respective binding assays.

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