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. Author manuscript; available in PMC: 2019 Mar 21.
Published in final edited form as: Science. 2014 Sep 19;345(6203):1512–1515. doi: 10.1126/science.1251550

Fig. 1. IRBIT interacts with RNR in a dATP-dependent manner.

Fig. 1.

(A) IRBIT-RNR interaction. IRBIT was immunoprecipitated from asynchronous HeLa cell lysates; bound proteins were resolved on SDS-PAGE and stained with Coomassie brilliant blue (CBB). IRBIT and R1 proteins are indicated by arrows. (B) Minimal domain required for dATP-dependent IRBIT-RNR interaction. GST-IRBIT64−87 was mixed with R1/R2B and 3 mM dATP. Complexes were retrieved by glutathione-Sepharose and resolved by SDS-PAGE and stained with CBB. (C) Alignment of IRBIT domain from different metazoans. (D) R1-IRBIT interaction is evolutionarily conserved. GST-IRBIT (hIRBIT) or GST-CG9977 (dIRBIT) were mixed with either human 6His-R1 (hR1) or Drosophila 6His-R1 (dR1) along with 3 mM dATP and analyzed as in (B). (E) hIRBIT binds Xenopus R1. GST-hIRBIT was mixed with cytostatic factor–arrested Xenopus laevis egg extracts. IRBIT-bound proteins were purified and probed with antibodies against R1 and Symplekin (4).