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Fig. 3. Mapping of the JIL-1 interaction domain with Chromator. (A) Diagrams of the JIL-1 and Chromator proteins indicating the domains to which GST-fusion proteins were made for mapping. In the overlay experiments various truncated JIL-1 GST-fusion protein constructs to the domains in A or a GST-only control were fractionated by SDS-PAGE, western blotted, incubated with Chro-CTD (B) or Chro-NTD (C) GST-fusion protein, and interactions detected with the C-terminal Chromator mAb 6H11 (B) or the N-terminal Chromator mAb 12H9 (C). The only interaction detected was between the JIL-1-CTD and Chro-CTD fusion proteins (arrows in B). Immunoblots of the overlay GST-fusion proteins Chro-CTD and Chro-NTD are shown (B, lane 7 and C, lane 6, respectively). (D) Immunoblot of the input GST-fusion proteins used for the overlay experiments in B and C detected with the anti-GST mAb 8C7. (E) Overlay experiments with truncated C-terminal JIL-1 GST-fusion protein constructs to the subdomains shown in A or a GST-only control were fractionated by SDS-PAGE, western blotted, incubated with Chro-CTD, and interactions detected with the C-terminal Chromator mAb 6H11. In these experiments interactions between Chro-CTD and JIL-1-CTD as well as JIL-1-CTD-A were detected (arrows) but not between Chro-CTD and JIL-1-CTD-B. An immunoblot of the overlay GST-fusion protein Chro-CTD is shown in lane 5. (F) Immunoblot of the input GST-fusion proteins used for the overlay experiments in E detected with the anti-GST mAb 8C7. This defined the JIL-1 C-terminal acidic domain as sufficient for mediating interactions with the C-terminal domain of Chromator. The relative migration of molecular size markers is indicated to the right of the immunoblots in kDa.

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