Activity and subcellular compartmentalization of peroxisome proliferator-activated receptor are altered by the centrosome-associated protein CAP350

JCS01600 Supplemental Material

Files in this Data Supplement:

• Movie 1 -

  Movie 1. (see Fig. 4A) CAP350 immunofluorescence. Three-dimensional voxel Z-stack and 60 degree rotation in 5 degree increments of a NIH3T3 cell transfected with c-Myc-tagged CAP350 and visualized by indirect immunofluorescence with an anti-human c-Myc antibody and an anti-mouse antibody conjugated to Texas Red.

• Movie 2 -

  Movie 2. (see Fig. 4B) CAP350 colocalization with PPARa. Three-dimensional voxel Z-stack and 60 degree rotation in 5 degree increments of a NIH3T3 cell transfected with c-Myc-tagged CAP350 and visualized by indirect immunofluorescence with an anti-human c-Myc antibody and an anti-mouse antibody conjugated to Texas Red. PPARa was visualized with an anti-PPARa antibody and an anti-rabbit antibody labeled with the dye Alexa Fluor 488 (green). The overlapping red/green signal is pseudocolored yellow.

• Movie 3 -

  Movie 3. (see Fig. 6B) CAP350 localizes to the centrosome. Three-dimensional voxel Z-stack and 60 degree rotation in 5 degree increments of a NIH3T3 cell transfected with c-Myc-tagged CAP350 and visualized by indirect immunofluorescence with an anti-human c-Myc antibody and an anti-mouse antibody conjugated to Texas Red. Pericentrin was visualized with an anti-pericentrin antibody and an anti-rabbit antibody labeled with the dye Alexa Fluor 488 (green). The overlapping red/green signal is pseudocolored yellow. Z-stacks were captured in 0.2 mm increments of 6-8 optical slices using a 60´ oil immersion plan apochromat objective (Nikon). All image stacks were digitally captured using Simple PCI v.5.2 (Compix, Cranberry Township, PA). Optical slices were deconvolved with Autodeblur v9.1 (Autoquant Imaging, Albany, NY). Three-dimensional voxel generations, rotations and MPEG videos were created using Imaris v4.0 (Bitplane Software, Zurich, Switzerland).