Figure 2.

Ubp10 co-assembles with SIR complex onto chromatin. A, schematic for testing Ubp10 binding to pre-assembled heterochromatin using the recombinant (recomb) proteins, SIR complex and Ubp10. SIR complex was pre-incubated with chromatin beads before the addition of Ubp10 to the reaction. B, Western blot analysis of pre-assembled heterochromatin pulldown. Ubp10 signal in mock reaction quantified as 1, and Ubp10 signal in +SIR complex sample is relative to mock. Mock and relative signals were normalized to H3. Proteins were detected using antibodies specific for epitope tags, GST-Ubp10, 3×HA-Sir2, and Sir3–3×FLAG, whereas H3 was detected by its native epitope. C, schematic for testing co-assembly of Ubp10 and SIR complex to chromatin using recombinant proteins, SIR complex and Ubp10. SIR complex was pre-incubated with Ubp10 before the addition of chromatin beads to the reaction. D, Western blot analysis of co-assembly of Ubp10 and SIR complex on chromatin, analyzed as in B.