DIAPH1 interaction partner identification using BioID.

293T cells were transfected with p TRIPZ BirA tagged DIAPH1. Two days post transfection, cells were treated with puromycin to select transfectants. The entire BirA-DIAPH1 cassette is under the control of Tet inducible promoter. Induction of DIAPH1 was achieved by treating cells with Doxycycline (10ug/ml) for 24hrs followed by 50uM of Biotin treatment, overnight, to induce biotinylation of interacting partners. Cells treated with Biotin alone (no Dox) were used as a control for IP. Cytoplasmic extracts were prepared and subjected to immunoprecipitation using Streptavidin magnetic beads. Beads were washed with RIPA buffer and subjected to Mass spectrometry based protein identification to find interacting partners of DIAPH1.