Fig. 1: USP27X interacts with CBX2.
From: Phosphorylation of USP27X by GSK3β maintains the stability and oncogenic functions of CBX2
A–C Cell lysates from MCF7 (A), MDA-MB-231 (B), and BT-549 (C) were analyzed by IP using antibodies against USP27X and CBX2, then subjected to IB analysis. IgG was used as the isotype control. D Triple immunofluorescence (IF) staining of USP27X(green), CBX2(red), and nuclei (DAPI, blue) was performed in MCF7, MDA-MB-231, and BT-549 cells. Scale bar, 10 μm. E Purified Myc-CBX2 was incubated with GST, GST-USP27X or GST-USP27X C87A coupled to glutathione-Sepharose beads. Proteins retained on Sepharose were then subjected to IB with indicated antibodies. Recombinant GST-USP27X and GST-USP27X C87A were purified from bacteria and analyzed by SDS-PAGE and Coomassie blue staining. F Representative images show merged PLA and nuclear (DAPI) channels from PLA experiments. In situ PLA between endogenous USP27X and CBX2. Each red dot represents detection of the USP27X-CBX2 interaction complex. Scale bar, 10 μm. G, H Schematic representation of HA-tagged full-length (FL) USP27X (G), Myc-tagged FL CBX2 (H), and their various deletion mutants. I, J HEK-293T cells were cotransfected with HA-USP27X and Myc-tagged FL CBX2 or its deletion mutants and anti-Myc or HA-coupled magnetic beads for IP analysis of cell lysates followed by IB analysis with HA and Myc antibodies. K, L HEK-293T cells were cotransfected with Myc-CBX2 and HA-tagged FL USP27X or its deletion mutants and anti-Myc or HA-coupled magnetic beads for IP analysis of cell lysates followed by IB analysis with HA and Myc antibodies. For all panels, data are representative results of three independent experiments.
