Figure 6: Physical interaction between SBNO1 and Yap/Tead4 or Notch/Rbpj.

(a) SBNO1-Myc, HA-Yap, and/or FLAG-Tead4 were co-expressed in 293 T cells, and immunoprecipitated with indicated antibodies (α-Myc, α-HA and α-FLAG, respectively). Interactions between HA-Yap and FLAG-Tead4, and SBNO1-Myc and FLAG-Tead4 were observed by co-precipitation. Expression of HA-Yap attenuated the interaction between SBNO1-Myc and FLAG-Tead4, as observed by faint bands of co-precipitation (red arrowheads). Myc-SBNO1 co-precipitated HA-Yap, whereas HA-Yap did not co-precipitate SBNO1-Myc. (b) SBNO1-V5, Myc-NICD1, and/or FLAG-Rbpj were co-expressed in 293 T cells, and immunoprecipitated with indicated antibodies (α-V5, α-Myc and α-FLAG, respectively). FLAG-Rbpj was co-immunoprecipitated with SBNO1-V5 (α-V5) and Myc-NICD1 (α-Myc), indicating their interaction. This was further confirmed by co-precipitation of SBNO1-V5 and Myc-NICD1 with FLAG-Rbpj (α-FLAG). The interaction between SBNO1 and Rbpj was weak in the presence of NICD1, as observed by faint bands (red arrowheads). (c–e) Interaction between Sbno1 or Yap and Tead4 in E3.5 embryos was analyzed by in situ PLA. Green signals indicate the ligated antibodies, which represent physical interaction of the antigens. Antibodies against Sbno1 or Yap with Tead4 gave clear signals in the nuclei of outer cells (d,e), while α-Tead4 antibody alone did not give any signal (c). Uncropped image of blots are shown in Supplementary Fig. 11.