FIG 1.

Biochemical characterization of SMCHD1 complexes. (A) Canonical SMC complex, represented here by cohesin, composed of an SMC1-SMC3 heterodimer and an Scc1 (kleisin) subunit. The SMC hinge (HD) and Walker A/B ATPase (A/B) are denoted. (B) Schematics illustrating the domain architecture of canonical SMC proteins and SMCHD1. GHKL ATPase (GHKL), coiled-coil (cc), and bromo-adjacent homology (BAH) domains are denoted. (C) Western blots showing expression of SMCHD1-FLAG or SMCHD1 in parental (PGK12.1) and stable (clone C6) cell lines used in mass spectrometry experiments. Arrowheads indicate full-length endogenous or FLAG-tagged SMCHD1 protein (227 kDa). The four lanes shown for each blot are the input (In), flowthrough (FT), and elutions 1 and 2 (E1 and E2, respectively) from anti-FLAG IP. SMCHD1 is present only in eluents from clone C6. (D) Silver staining of IP material for mass spectrometry from control (PGK) and clone C6 nuclear extracts. (E) Western blot of fractions following size exclusion chromatography of nuclear extracts from C6 clone for FLAG-tagged SMCHD1 (top) and from PGK12.1 ESCs for endogenous SMCHD1 (bottom). (F and G) Western blots of fractions generated from sucrose gradient separation of nuclear extracts from the C6 clone (anti-FLAG) and PGK12.1 (anti-SMCHD1), for all fractions (F) and only selected fractions (G). Molecular mass standards (in kilodaltons) for panels E to G are labeled above each blot.