Fig. 5. Increased proteasome activity and reversibility of vimentin aggregates in vivo. (A,B) Immunofluorescence analysis of lenses from 4-month-old VimR₁₁₃C transgenic and wt mice, stained with antibodies against total (green) and transgenic vimentin (red). Panels 1-3 show higher magnification of lens areas that represent different stages of fibre cell differentiation. Notice that towards the centre of the lens that contains the oldest fibre cells, vimentin aggregates become dissolved, suggesting that they are transient. (C) Fluorescence-based proteasome assay of extracts from lenses and from cells stably transfected with VimR₁₁₃C. In both settings, a 30% increase in proteasome activity in the mutant, compared with the wt, was detected. (D) SDS-PAGE and immunoblotting for ubiquitin of VimR₁₁₃C and wt lens extracts after immunoprecipitation with a vimentin antibody. Substantially increased ubiquitylation of vimentin was detected in the transgenic lenses (indicated by arrows). The position of the Ig heavy chain is indicated. (E) Immunoblot for Hsp70 in soluble and insoluble lens extracts of wt and VimR₁₁₃C transgenic mice. In both fractions, Hsp70 expression was upregulated in VimR₁₁₃C lenses. Scale Bars: 100 µm (A,B), 15 µm (panels 1-3).