List of primers used in cloning and qPCR.

Identification of protein-protein interactions is essential for understanding protein functions in biological processes. While immunoprecipitation has traditionally been used to isolate proteins and their partners, it faces limitations in capturing transient interactions. Proximity labeling, particularly with the biotin ligase TurboID, addresses this challenge by enabling rapid and efficient identification of interacting proteins in vivo. Human induced pluripotent stem cells are valuable models for studying human development, however certain biological processes, such as differentiation, can be difficult to analyze because conventional transfection methods are challenging. Therefore, an alternative strategy for detection of interacting proteins is necessary. Here, we developed a novel system employing TurboID-fusion proteins within an integrative and inducible expression vector to investigate the interactome during stem cell differentiation. We validated our system by using U2AF2 and GFP as bait proteins, generated two distinct cell lines, and determining the minimum induction time required for optimal protein expression. Our results confirmed that the system did not alter the expected localization of U2AF2. Applying our system, we identified significant differences in the interactome of U2AF2 between the pluripotent and mesodermal differentiation stages, demonstrating that U2AF2 interacts with distinct protein sets following cell fate commitment. Our study successfully unveils a new tool for studying protein-protein interaction in human stem cells.