NA14 localizes to the centrosome and interacts with the M87 isoform of spastin.

<p>(A) Top, Schematic diagrams showing the domain organizations of spastin isoforms generated through use of 2 different translation start codons (exon 4 splice cassettes are also indicated). Bottom, HeLa cells were transfected with Myc-tagged spastin M1 or spastin M87 and immunoblotted (IB) with Myc-tag antibody. Migrations of molecular weight standards (in kDa) are indicated at the left. (B) Myc-spastin isoforms M1 and M87 (green) were co-stained with pericentrin antibodies (red) and visualized by confocal microscopy. Scale bars: 10 µm. (C) Myc-spastin isoforms M1 and M87 (red) were co-stained with pericentrin antibodies (green) and visualized by confocal microscopy. Relative fluorescence intensities for the indicated linear regions in merged images (boxed areas enlarged in insets at the bottom left) were measured using Zeiss LSM710 software and graphed (right). Scale bar: 10 µm. (D) Relative numbers of HeLa cells accumulating the indicated endogenous or recombinant spastin proteins in the centrosome were quantified (means ±SEM; <i>n</i> = 3, with 100 cells per trial). (E) HeLa cells were transfected with Myc–spastin M1 or M87 as indicated, immunoprecipitated (IP) with NA14 antibodies or non-immune IgG, and immunoblotted (IB) with Myc-epitope antibodies. The input represents 5% of the starting material. Migrations of molecular weight standards (in kDa) are indicated at the left, and the spastin isoform to the right.