Figure 3

VB3 and VB6 chromobodies recognize vimentin in living cells.

(a) Intracellular immunoprecipitation (IC-IP) of vimentin. Lysates of HEK293T cells expressing indicated chromobodies (VB3-CB; VB6-CB) or GFP were subjected to immunoprecipitation with the GFP-Trap. Input (I) and bound fractions (B) were analyzed by immunoblot with an α-VIM-IgG (upper panel) and an anti-GFP antibody (lower panel). (b,c) Vimentin chromobodies have a low tendency to aggregate upon intracellular expression. (b) Representative images of HeLa cells expressing GFP-VIM, VB3-CB or VB6-CB from three independent experiments. Scale bar: 20 μm. (c) Quantification of cells with fluorescent aggregates upon expression of GFP-VIM, VB3-CB or VB6-CB. Columns represent the percentage of cells displaying fluorescent granules (total number of analyzed cells > 300). Values represent the means of three independent transfections ± stds. For statistical analysis Chi-squared test was used, ***P < 0.001. (d) Fluorescent recovery after photobleaching (FRAP) analysis of VB3-CB, VB6-CB and GFP-VIM. Shown are representative images of transiently transfected HeLa cells before and after photobleaching of a defined region (white box). Scale bars: 10 μm. (e) Quantitative evaluation of FRAP data showing mean values of fluorescence recovery in photobleached regions. VB6-CB recovered to 84.1 ± 3.1% with a halftime of 3.9 s, the recovery of VB3-CB amounted to 58.4 ± 19.5% with a half time of 4.3 s; n = 10; N = 1. Data are represented as mean ± stds. For statistical analysis students t-test was used, ***P < 0.001.