Figure 14

Probing of a commercial protein microarray revealed strong binding of the patient's sera to human ARHGAP26 (Panel A). In accordance with this finding, binding of IgG from the patient's serum (Lane P), but not from three healthy controls (Lane C1-C3), to a recombinant human full length ARHGAP26 protein was found (Panel B). Western blotting confirmed the presence of ARHGAP26 in primate cerebellar extract (Panel C, lane 2), and incubation with the patient's CSF resulted in a band running at the same height as the ARHGAP26 band (Panel C, lane 1); the significance of the additional band recognized by the commercial antibody is unknown. The commercial ARHGAP26 antibody bound to the Purkinje cell layer and the molecular layer (Panel D) and showed a good overlay with the patient's IgG depicted in yellow (Panel E). Finally, preadsorption of the patient's CSF with the ARHGAP26 protein (Panel H), but not preadsorption with a control protein (Panel G), resulted in complete loss of binding to cerebellar tissue sections in an indirect immunofluorescence assay; panel F shows binding of IgG from a non-preadsorbed aliquot of the same CSF sample (exposure time was 2 sec in all cases to detect also low fluorescence signals).