Co-immuno-precipitation of Strip2

To identify the interaction partners of Strip2, we generated an ESC cell line permanently overexpressing Strip2 in undifferentiated wild-type ESCs. Briefly, generation of the Strip2-Turbo-GFP-fusion overexpressing wild-type ESCs (next referred transient Strip2 overexpressing ESCs) was performed by the Magnetofection^TM transfection technology using a plasmid with the cDNA cassette encoding for Strip2 and the DNA cassettes for GPF fusion construct and puromycin resistant cassette (Catalogue number: {"type":"entrez-nucleotide","attrs":{"text":"MG213986","term_id":"1319665384","term_text":"MG213986"}}MG213986, pCMV6-AC-GFP; OriGene), as mentioned above. In parallel, a control ESC cell line was generated using a control plasmid without the Strip2 cDNA but with the eGFP and neomycin cassette (next referred as GFP-F + ESCs). Transient Strip2 overexpressing ESCs were passaged for five times under standard cell culture conditions (37 °C, 5% CO₂). To identify only specific interactions of Strip2 with other proteins, GFP-F + ESCs were used in parallel. Protein extraction and IP was performed using the ChromoTek TurboGFP-Trap Agarose Kit (tbtak-20; Proteintech, Germany), according to the instructions of the kit. The cell extract was prepared using RIPA buffer (Thermo Fisher Scientific). The cell lysate was mixed with TurboGFP-Trap-A beads and equilibrated for 1 h at 4 °C using an end-over-end shaker. After incubation, the samples were washed three times, and then the specific Strip2 bound proteins were eluted using a buffer. The eluted proteins were further analyzed by mass spectrometry.