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. 2001 Mar 5;152(5):877–894. doi: 10.1083/jcb.152.5.877

Figure 2.

Figure 2

FTCD binds directly to vimentin IFs. (A) FTCD expression was induced by IPTG in bacteria, and FTCD was purified as described in Materials and Methods. A Coomassie blue–stained gel shows a major 58-kD FTCD band (arrowhead). (B) Bacterially expressed and purified FTCD was mixed in different ratios with polymerized vimentin filaments and centrifuged at 100,000 g for 30 min at 20°C. The supernatants and pellets were recovered, and an equivalent proportion of each was processed by SDS-PAGE. The proteins were transferred to NC and immunoblotted with polyclonal anti-FTCD antibodies. FTCD remains soluble in the absence of vimentin filaments (lanes 5 and 6), but pellets when vimentin filaments are added (lanes 1 to 4). In lanes 7 and 8, only vimentin without added FTCD was processed. (C) BSA was incubated with vimentin filaments, and the mixture was centrifuged as above. The supernatant and pellet were recovered, and an equivalent proportion of each was processed by SDS-PAGE and Coomassie blue staining. BSA does not bind to and is not recovered with vimentin filaments. (D–G) Vimentin filaments alone (Vimentin) or incubated with bacterially expressed and purified FTCD (Vimentin + FTCD) were stained with uranyl acetate and visualized by EM. In the vimentin only sample, the filaments appear smooth (D), whereas filaments incubated with FTCD are decorated with spherical structures (E). A higher magnification of the area in E, is shown in F. The size of the spherical structures is ∼12 nm (arrows) and is consistent with the size of FTCD octomers. The spherical structures were often seen linking individual vimentin filaments (G, arrows). (H) rFTCD–pBS was used as template in an in vitro transcription/translation reaction. A fluorograph of generated products shows a major 58-kD protein (arrowhead). (I) Bacterially expressed and purified FTCD, BSA and bacterially expressed and purified mouse vimentin were processed by SDS-PAGE, and the gel stained with Coomassie blue to show relative amounts of proteins. (J) A gel analogous to that in I was transferred to NC and overlaid with 35S-FTCD, as generated in H. Radiolabeled FTCD binds directly and specifically to FTCD and vimentin. Bar, 100 nm.