Identification of mitotic substrates of phospho-regulated USP9X

We previously identified the deubiquitylase USP9X as a G2/M-specific CDC14B interactor (PXD012732) and subsequently identified USP9X as a specific CDC14B target (PXD012733). To investigate relevant mitotic substrates of phospho-regulated USP9X, we next performed a Stable isotope labeling by amino acids in cell culture (SILAC)-based screen in which ubiquitylated proteins were purified from control or USP9X depleted HEK 293T cells that were either asynchronous or synchronized in mitosis. Stable expression of recombinant His-tagged ubiquitin has been used for selective enrichment of ubiquitinylated proteins in both conditions.