Figure 7.

Association of PP2A with vimentin in Hs68 fibroblasts. (A and B) Vimentin was isolated from Hs68 fibroblasts by Triton X-100 and 0.6 M KCl extraction as described in MATERIALS AND METHODS. Material corresponding to one-third of a 100-mm dish of Hs68 was electrophoretically analyzed; equal proportions of the soluble (lanes S; S/10, of S) and insoluble, vimentin-enriched (lanes VE) fractions were electrophoretically separated. Subsequently, proteins were stained with Coomassie brilliant blue (A). Arrows indicate the positions of vimentin (v) and actin (a). The migration of molecular mass markers is indicated at the left. Alternatively, gels were immunoblotted (B) for the presence of PP2Ac and PR65 using AbC^302/309 (upper panel) and Ab65^177/196 (middle panel). Approximately 5 ng of rabbit skeletal muscle PP2Ac and ∼10 ng of recombinant PR65 were included as a positive control (lanes C), and their positions are marked with arrowheads.Because of a high background staining in the region at ∼50 kDa, B55 could not be detected in an unequivocal way using our polyclonal Ab55. Vimentin was isolated from Hs68, transiently transfected to express HA-tagged B55. These preparations were immunoblotted using the monoclonal anti-HA (12CA5) (lower panel). nt, vimentin from nontransfected cells; t, from transfected cells. The arrowheads indicate the migration of HA-B55 as determined by anti-HA immunoprecipitates, run on the same gel and detected with Ab55^recomb. (C) Hs68 fibroblasts were grown on glass coverslips and, before formalin fixation, extracted with 0.01% Triton X-100. Subsequently, cells were stained for PP2Ac, PR65, or B55 using AbC^recomb, Ab65^177/196, or Ab55^recomb, respectively, and costained for vimentin. The staining for PP2A subunits was revealed using FITC-conjugated reagents (upper panels), whereas vimentin was detected using Texas Red (middle panels). The lower panels show double exposures of the respective PP2A and vimentin staining (see MATERIALS AND METHODS). Bar, 5 μm.