SYT-SSX2 Antagonizes Polycomb Figure 1. SYT-SSX2 associates with Polycomb complex proteins. (A) Equivalent protein levels of retrovirally expressed SYT-SSX2 (FLAG and HA-tagged) in U2-OS cells and in a primary human synovial sarcoma cell line (Syn1). 100 ug of cellular lysate-derived protein was loaded in each lane. SYT-SSX2 was visualized with the anti-FLAG tag antibody in U2-OS and with a SYT-specific antibody [SV11; 37] in Syn1 cells. (B) Colocalization of SYT- SSX2 with Bmi1 and Ring1B. Cells infected with pOZ viral vector, SYT, SYT-SSX2, SYTdel8, and SSX2 (all FLAG and HA-tagged), were analyzed by indirect immunofluorescence. Infected cDNAs (HA, red) and Bmi1 or Ring1B (green) were visualized individually and with merging of the two channels (merge). The cytoplasmic HA staining in pOZ-infected cells is due to the generation of an irrelevant FLAG/HA-tagged peptide by the vector. Image magnification was at 636. (C) Decrease in Bmi1 fluorescence in SYT-SSX2 expressing cells. U2OS cells infected with SYT-SSX2 were analyzed by indirect immunofluorescence for SYT-SSX2 (HA; red) and Bmi (green). Image magnification was at 636. Average fluorescence (20 cells per replicate; n = 3) of Bmi1 was compared between SYT-SSX2-infectants and uninfected cells using MetaMorph software and plotted. (D) In vitro binding of Ring1B to SYT-SSX2. Upper panel: GST, GST-Bmi1 and GST-SYT-SSX2 visualized by Coomassie staining. The asterisks (*) indicate full-length proteins. Lower panel: autoradiography of in vitro-translated (IVT) Ring1B bound to GST-Bmi1 (positive control) and GST-SYT-SSX2. Lane 1 represents 10% of input IVT Ring1B. doi:10.1371/journal.pone.0005060.g001 PLoS ONE | www.plosone.org 3 April 2009 | Volume 4 | Issue 4 | e5060