FIG. 1.

BCOR and NSPC1 are obligate partners in an ∼800-kDa complex. (A) Western blotting and silver stain analysis of size-exclusion fractionation (Superose 6) of the BCOR complex purified from a tagged BCOR HeLa S3 cell line. Bands at 20 kDa, 25 kDa, 32 kDa, 35 kDa, 50 kDa, and 70 kDa and tagged BCOR elute as a tightly migrating ∼800-kDa complex but at a 2% yield. (B) Silver stain analysis and protein identifications from purifications of HEK293 cells stably expressing untagged GFP (Mock), TAP-Flag (FL)-tagged BCOR, or TAP-FL-tagged NSPC1. Bands immediately below BCOR and at 40 kDa were identified as BCOR and tagged NSPC1 degradation products, respectively. All identifications were confirmed by tandem mass spectrometry fragmentation except for the four observed peptides from RYBP, which were not amenable to fragmentation. HSP70 has been observed in other PcG complexes (46). The band at 24 kDa (•) was present in both purifications, but insufficient data prevented definitive identification. (C) Schematic diagram of proteins found in the BCOR complex. The amino terminus of FBXL10 (shown in gray) containing the JmjC domain was not detected by mass spectrometry. RYBP has fewer amino acids than NSPC1 but has been shown to run aberrantly at 32 kDa in previous studies (30). The following abbreviations were used: A, ankyrin; C, CXXC; P, PHD; F, F-box; L, leucine-rich repeat; ZF, zinc finger. Two alternatively spliced isoforms of BCOR (A and C) used in this study differ by 34 amino acids.