Whole-cell extracts were prepared and subjected to Western blotting. IgG Sepharose was used to isolate Cps60/Bre2 from nuclear extracts. Immunoprecipitations were assayed by Western blotting with antibodies to Set1, the Flag epitope on Set1, or the TAP tag on Cps60/Bre2. TAP purification of Cps60 was performed as previously described (Miller et al. 2001 (link); Takahashi et al. 2011 (link)).
For LEO1 complementation, cells containing the pRS315 vector bearing LEO1 under the control of the GAL1 promoter were cultured in SD medium deficient of leucine overnight at 30°C, transferred to synthetic medium containing dextrose and galactose, respectively, and incubated for 6 h at 30°C. Cell extracts were prepared and analyzed by Western blotting. Antibodies were generated by our laboratory except for anti-H3K79me3 (Abcam, ab2621), anti-monoubiquitinated histone H2B (Cell Signaling Technology, #5546), anti-TAP (Thermo Scientific, CAB1001), and the M2 anti-Flag antibody (Sigma, F3165).
For LEO1 complementation, cells containing the pRS315 vector bearing LEO1 under the control of the GAL1 promoter were cultured in SD medium deficient of leucine overnight at 30°C, transferred to synthetic medium containing dextrose and galactose, respectively, and incubated for 6 h at 30°C. Cell extracts were prepared and analyzed by Western blotting. Antibodies were generated by our laboratory except for anti-H3K79me3 (Abcam, ab2621), anti-monoubiquitinated histone H2B (Cell Signaling Technology, #5546), anti-TAP (Thermo Scientific, CAB1001), and the M2 anti-Flag antibody (Sigma, F3165).