Fig. 4

Identification of Rho-associated protein kinase 2 as the target antigen. a Lysates of rat cerebellum were incubated with patient or control sera (1:33). Immunocomplexes were isolated with protein-G-coated magnetic beads, eluted by SDS and subjected to SDS-PAGE analysis followed by (left) staining with colloidal Coomassie, (middle) Western blot using patient serum or (right) Western blot using polyclonal rabbit anti-ROCK2 and Ponceau S staining. Arrow indicates the position of the immunoprecipitated antigen at about 160 kDa. b Double immunofluorescence staining of cerebellar tissues with patient serum (1:50, green) and rabbit anti-ROCK2 antibody (1:250, red). The anti-ROCK2 antibody produced fluorescence patterns matching those generated by the patient’s serum. Scale bar = 100 μm ML = molecular layer, PL = Purkinje cell layer, GL = granule cell layer