Figure 2

Interaction partners of TIPT2 and geminin (Gmnn). (A) Binding of His-geminin to an array of 63 peptides with 17 amino acids overlapping 20-mers derived from the TIPT2 sequence. TIPT2 peptides bound specifically to His-geminin as detected by anti-geminin antibodies. KRKK amino acids of TIPT2 were mapped as the geminin-binding domain. (B-G) In vitro pull-down assays were performed using purified GST-TIPT2, GST-TIPT2m (containing a mutated geminin-binding site), GST-Gmnn or GST as bait. As preys were used in vitro transcribed/translated proteins (B-D), mouse adult testis (F) and human U2OS cell extracts (G). For experimental details please see methods section. (B) GST-TIPT2, GST-TIPT2m or GST interaction with radioactively labelled geminin. (C) GST-TIPT2 binding to in vitro translated Scmh1, Mph2, Ring1B, TBPL1, TBP, TFIIB, TIPT2 and Mel18. (D) GST-Gmnn interaction with in vitro translated TBP, TBPL1 and TFIIB. (E) Ponceau staining of GST proteins used for the protein lysate pull-down assays. (F) GST-TIPT2 and GST-Gmnn interaction with endogenous TBP, geminin and TIPT2 from adult mouse testis extract. (G) GST-TIPT2 and GST-Gmnn interaction with endogenous TBP from U2OS cell extract. The star indicates the cross-reactivity of TBP antibodies raised against GST recombinant human TBP with GST-TIPT2. (H) Co-immunoprecipitation of endogenous TBP with HA-TIPT2 using anti-HA antibodies. Extracts from untransfected U2OS cells, and from a U2OS clone stably producing HA-TIPT2 (HA-TIPT8), respectively, were analyzed.