Fig. 9

Torsin1 is a neuronal glycoprotein.

A: The peptide sequence used to raise polyclonal antibodies to zebrafish Torsin1 is shown underlined in bold. An alignment between Torsin1 and the homologous regions of other zebrafish Torsin-like proteins is shown to illustrate the lack of potentially cross-reacting sequences in other Torsins. Amino acids that are identical to Torsin1 at each position are highlighted in yellow. B: HEK cells were transfected with a plasmid encoding Torsin1b (lane 1) or with empty vector (lane 2). A western blot made with lysates from transfected cells was probed with affinity-purified Torsin1 antibody. The blot was then re-probed with an antibody to β-actin as a control for equal protein loading (lower panel). C: Identical samples of zebrafish whole brain lysate were resolved by SDS-PAGE in adjacent lanes of a gel and the resulting western blot was divided into strips each containing a single lane. The strips were probed with either affinity-purified Torsin1 antibody (lane 1), affinity-purified Torsin1 antibody pre-incubated with the peptide immunogen shown in panel A (lane 2), or pre-immune serum (lane 3). The blots were then re-probed with an antibody to β-actin as a control for equal protein loading (lower panel). D: Whole brain lysate was either treated with PNGase F (lane 2) to remove N-linked carbohydrate moieties, or mock treated without addition of PNGase F (lane 1), prior to SDS-PAGE and immunoblotting with Torsin1 antibody. E: Schematic representation of Torsin1 showing the positions of the consensus N-glycosylation sites. F–K: Adult zebrafish CNS sections were labeled with Torsin1 antibody; immunoreactive structures were revealed by chromogenic histochemistry with a red reaction product. Sections were counterstained with Mayer’s hematoxylin so that nuclei appear blue. (F) Parasagittal sections of the whole zebrafish brain are shown labeled with Torsin1 antibody. In the lower panel, the antibody was pre-incubated with the peptide immunogen. (G) Optic tectum. Key: SM, stratum marginale; SO, stratum opticum; SFGS, stratum fibrosum griseum superficiale; SGC, stratum griseum centrale; SAC, stratum album central; SPV, stratum periventriculare. (H) Periventricular layer of optic tectum (upper panel) and medulla oblongata (lower image), showing cytoplasmic Torsin1 expression in neurons (arrowheads). (J) Thalamus (upper panel) and cerebellum (lower panel) showing Torsin1-related immunoreactivity in neuropil; arrowhead in upper panel shows a neuron surrounded by punctate Torsin1 immunoreactivity that may be present in nerve terminals. (K) Spinal cord at low magnification (upper panel) and high magnification (lower panel) showing robust Torsin1 immunoreactive signal in gray matter, but absence of Torsin1 in white matter. The arrowhead in the lower panel shows the myelin sheath surrounding the Mauthner axon (MA).