Fig. 4. TES interacts with adhesion proteins. (A) Rat-1 fibroblasts stably overexpressing full-length GFP-tagged TES were stained with mouse zyxin hybridoma supernatant (164D4) and TRITC-labelled mouse secondary antibody to visualise endogenous zyxin. (B) Rat-1 fibroblasts stably expressing full-length GFP-tagged TES were stained with anti-mena antibody and TRITC-labelled mouse secondary antibody to visualise endogenous mena. (C) HeLa WCE incubated with GST protein alone or various GST-tagged TES constructs are shown. Complexes were separated by 7.5% SDS-PAGE and blots probed with an anti-zyxin antibody. (D) GST-alone or GST-tagged TES proteins were incubated with in-vitro-transcribed/translated zyxin extract. Complexes were separated by 7.5% SDS-PAGE and blots probed with anti-zyxin antibody. HeLa WCE were incubated with GST protein alone or GST-tagged full-length TES and complexes separeated using 7.5% SDS-PAGE before transfer to a PVDF membrane and probing with (E) an anti-mena antibody, (F) an anti-VASP antibody, (G) anti-talin antibody or (H) an anti-actin antibody. (I) GST and GST-tagged proteins used in the pull-down assays were run on 10% SDS-PAGE and stained with Coomassie brilliant blue to visualise protein bands. Positions of molecular weight markers (Amersham) are shown. GST, GST control; FL, GST-tagged full-length TES; LL, GST-tagged LIM-less TES; LIM1, GST-tagged 1st LIM domain; LIM1/2, GST-tagged 1st and 2nd LIM domains; LIM3, GST-tagged 3rd LIM domain; I, input (10% of input WCE was run alongside as a comparison). B, bound protein; U, unbound.

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