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. 2000 Jun 15;20(12):4596–4605. doi: 10.1523/JNEUROSCI.20-12-04596.2000

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Fig. 6. — Fish directly associates with POU and PAS domain proteins. A, Interactions between Fish, Sim, and Drifter revealed via yeast 2-hybrid assays. The following combinations of bait and prey constructs were transformed into yeast and assayed for growth on Leu^- medium: (1) Fish/TrhbHLH-PAS; (2) Fish/PerL; (3) Fish/Per; (4) Fish/Sim; (5) Fish/SimPAS; (6) Fish/SimbHLH; (7) Fish/SimbHLH-PAS; (8) Fish/SimbHLH-PASA; (9) Fish/Fish; (10) Fish/Drifter; (11) Fish/DrifterPOU; (12) Fish/Pdm-1; (13) Fish/Pdm-2; (14) Fish/Oct-3; (15) Drifter/Sim; (16) Drifter/SimbHLH-PAS; (17) DrifterPOU/Sim; (18) DrifterPOU/SimbHLH-PAS. Note that interactions were detected between Fish and the POU domain of Drifter, as well as the PAS domain but not the bHLH region of Sim. No interactions were detected between any Sim and Drifter constructs. B, Binding of GST–Fish fusion protein to ³⁵S-labeled Sim generated via in vitro translation. Lane 1, In vitro-translated Sim analyzed via SDS-PAGE and autoradiography. Note a radiolabeled band at 73 kDa corresponding to full-length Sim protein, as well as a second band of lower molecular weight.Lane 2, In vitro-translated Sim does not bind GST. Sim protein was incubated with GST and glutathione-Sepharose resin. The mixture was washed, and the bound material was eluted and analyzed via SDS-PAGE and autoradiography. Note the absence of labeled Sim protein. Lane 3, In vitro-translated Sim binds to GST–Fish. Sim protein was incubated with GST–Fish and glutathione-Sepharose resin. The mixture was washed, and the bound material was eluted and analyzed via SDS-PAGE and autoradiography. Note the presence of labeled Sim proteins. C, Binding of GST–Fish fusion protein to ³⁵S-labeled Fish generated viain vitro translation. Lane 1, In vitro-translated Fish analyzed via SDS-PAGE and autoradiography. Note a major radiolabeled band at ∼45 kDa corresponding to full-length Fish. Lane 2, In vitro-translated Fish does not bind efficiently to GST. Fish protein was incubated with GST and glutathione-Sepharose resin. The mixture was washed, and the bound material was eluted and analyzed via SDS-PAGE and autoradiography. Note the absence of labeled Fish protein.Lane 3, In vitro-translated Fish does bind GST–Fish. Fish protein was incubated with GST–Fish and glutathione-Sepharose resin. The mixture was washed, and the bound material was eluted and analyzed via SDS-PAGE and autoradiography. Note the presence of labeled Fish protein.