On 2016 Feb 16, Ditte Caroline Andersen commented:

Dear Dr. Bakker, Thanks for your interest in our study. The fusion protein generated by the pACT vector encompasses a strong nuclear localization signal of the SV40 large T antigen, driving fusion proteins into the nucleus. Omitting the membrane signal peptide has been suggested to improve nuclear localization of the fusion proteins by Waller and co-workers, but they did not observe an increase in relative luciferase activity after removing membrane-targeting sequences (Waller et al, 2010). In our study, we verified nucleus localization of the proteins using immunohistochemistry, and the conclusion is that DLK1 and NOTCH1 in that milieu do interact. Whether this also occurs at the membrane remains to be determined, but results herein as well as those reported by others suggest that this may be the case since DLK1 seems to have an impact on Notch signaling. Waller H, Chatterji U, Gallay P, Parkinson T, Targett-Adams P (2010) The use of AlphaLISA technology to detect interaction between hepatitis C virus-encoded NS5A and cyclophilin A. Journal of virological methods 165: 202-210

On 2016 Feb 15, Hans Bakker commented:

Can the authors explain how the mammalian two-hybrid system should work with proteins that are on the cell membrane?

On 2016 Feb 15, Hans Bakker commented:

Can the authors explain how the mammalian two-hybrid system should work with proteins that are on the cell membrane?

On 2016 Feb 16, Ditte Caroline Andersen commented:

Dear Dr. Bakker, Thanks for your interest in our study. The fusion protein generated by the pACT vector encompasses a strong nuclear localization signal of the SV40 large T antigen, driving fusion proteins into the nucleus. Omitting the membrane signal peptide has been suggested to improve nuclear localization of the fusion proteins by Waller and co-workers, but they did not observe an increase in relative luciferase activity after removing membrane-targeting sequences (Waller et al, 2010). In our study, we verified nucleus localization of the proteins using immunohistochemistry, and the conclusion is that DLK1 and NOTCH1 in that milieu do interact. Whether this also occurs at the membrane remains to be determined, but results herein as well as those reported by others suggest that this may be the case since DLK1 seems to have an impact on Notch signaling. Waller H, Chatterji U, Gallay P, Parkinson T, Targett-Adams P (2010) The use of AlphaLISA technology to detect interaction between hepatitis C virus-encoded NS5A and cyclophilin A. Journal of virological methods 165: 202-210