Figure 2
From: Real-time analysis of epithelial-mesenchymal transition using fluorescent single-domain antibodies
Nanobodies specifically recognize vimentin.
(a) Immunofluorescence staining of endogenous vimentin in fixed MDCK cells with VB3ATTO488, VB6ATTO488 and VB6-VB6ATTO488 in comparison to α-VIM-IgG. Shown are respresentative images from three independent experiments. Scale bar: 20 μm. (b) Immunoblot analysis of protein lysates derived from HeLa, HEK293T, MDCK or A549 cells using VB6-VB6ATTO488 for direct detection or an α-VIM-IgG as control. (c) Immunoprecipitation of endogenous vimentin with immobilized Nbs. Input and bound fractions of indicated cell lysates were analyzed by immunoblot with an α-VIM-IgG. ctr: pulldown with non-related nanobody. (d,e) VB3 and VB6 bind to the rod domain of vimentin (d) Schematic overview of vimentin domains fused to GFP. (e) HEK293T cells were transfected with the indicated constructs and subjected to immunoprecipitation with immobilized Nbs followed by immunoblot analysis of input and bound fractions with an anti-GFP antibody. ctr: pulldown with non-related nanobody.
