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. 2014 Nov 25;5:5548. doi: 10.1038/ncomms6548

Figure 5. ERG interacts with tubulin.

Figure 5

(a) Co-immunoprecipitation (IP) of purified cytoplasmic or nuclear cell fractions using an anti-ERG or anti α-tubulin antibody followed by immunoblot for the indicated proteins in DU145-ERG cells or in VCaP cells. For all experiments isotype-matched IgGs were used to control for non-specific binding to the beads used for immunoprecipitation. (b) Graphic representation of the full-length TMPRSS2-ERG encoded protein with the different domains colour coded as follows (green, N terminus (ΔN); red, pointed domain (ΔP); blue, the middle amino acids that correspond to the CAE/CD domain (ΔM); pink, the ETS domain (ΔE); gold, C terminal transactivating domain (ΔC)). (c) Co-immunoprecipitation (IP) of whole-cell extracts from DU145-GFP, 72 h following transient transfection with the Flag-tagged wild type or indicated deletion ERG constructs using either anti-Flag or anti-ERG antibody followed by immunoblot for the indicated proteins. Isotype-matched IgGs were used to control for non-specific binding to the beads used for immunoprecipitation. Immunoblot of input lysate for each transfection are shown to the left of each IP blot. (d) Microtubule co-sedimentation assay of DU145 cells transiently expressing ERG-WT (wt) or Vcap cells expressing endogenous ERG protein. One mg of precleared cell lysate from each condition (HSS) was supplemented with exogenous purified tubulin and subjected to a cycle of polymerization in presence of GTP and paclitaxel at 37 °C. The samples were centrifuged at 100,000 × g to separate the microtubule polymers (WP) from the soluble tubulin dimers (WS), resolved by SDS–PAGE and immunoblotted for ERG and tubulin. HSP=high-speed pellet (containing cellular debris and nonspecific protein aggregates); HSS=high-speed supernatant (precleared cell lysate); WP=warm pellet containing microtubule polymers; WS=warm supernatant containing soluble tubulin dimers. (e) Cell viability assay of the indicated DU145 cells transiently expressing each of the ERG deletion mutants as in (c) in response to 48 h treatment with cabazitaxel. Western blot images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 12.