Figure 2. Both CENP‐C^CR and CENP‐C^motif bind specifically to the CENP‐A nucleosome, and CENP‐C^CR easily competes out CENP‐C^motif bound to CENP‐A.

1. Schematic diagram of the full‐length CENP‐C protein, indicating parts involved in interactions with other proteins or homo‐dimerization. Constructs used in this study are depicted below the diagram.
2. Native PAGE gel stained with Coomassie blue showing complexes formed between CENP‐A or H3 nucleosome and CENP‐C^CR. Lane 1: CENP‐A nucleosome, Lanes 2–4: Increasing amounts of CENP‐C^CR are added to CENP‐A nucleosome. Generation of a sharp band with slower mobility indicates formation of a specific CENP‐A/CENP‐C^CR complex. Lane 5: H3 nucleosome. Lanes 6–8: Increasing amounts of CENP‐C^CR are added to H3 nucleosome. Smear on the gel indicates formation of non‐specific H3/CENP‐C^CR complexes.
3. Same experiment as in (B) using CENP‐C^motif. Lane 1: CENP‐A nucleosome. Lanes 2–4: Increasing amounts of CENP‐C^motif are added to CENP‐A nucleosome. Upon binding CENP‐C^motif, CENP‐A nucleosome migrates slower through the gel. Note only modest change in mobility due to small size of CENP‐C^motif, comparing to CENP‐C^CR in (B). Lane 5: H3 nucleosome. Lanes 6–8: Increasing amounts of CENP‐C^motif are added to H3 nucleosome. Smear on the gel indicates formation of non‐specific H3/CENP‐C^motif complexes.
4. Native gel showing CENP‐C^CR competing out CENP‐C^motif bound to CENP‐A nucleosome. Lane 1: CENP‐A nucleosome. Lane 2: CENP‐A/CENP‐C^motif complex. Lane 3–6: Increasing amounts of CENP‐C^CR are added to the pre‐formed CENP‐A/CENP‐C^motif complex. Formation of slower migrating bands indicates that longer CENP‐C^CR is replacing shorter CENP‐C^motif bound to the CENP‐A nucleosome.
5. Native gel showing the inability of CENP‐C^motif to compete out CENP‐C^CR bound to CENP‐A nucleosome. Lane 1: CENP‐A nucleosome. Lanes 2 and 6: CENP‐C^CR/CENP‐A nucleosome complex. Lanes 3–5: Increasing amounts of CENP‐C^motif are added to the pre‐formed CENP‐A/CENP‐C^CR complex. Formation of smear at high amounts of CENP‐C^motif added indicates that CENP‐C^motif, at high concentrations, non‐specifically binds CENP‐A/CENP‐C^CR complex rather than replacing bound CENP‐C^CR. Lanes 7–9: Increasing amounts of CENP‐C^CR‐short are added to the pre‐formed CENP‐A/CENP‐C^CR complex. Formation of bands with higher mobility indicates that smaller CENP‐C^CR‐short is effectively replacing bigger CENP‐C^CR bound to CENP‐A nucleosome.

Data information: For (B–E), 2.4 μM nucleosomes are used in all experiments.