Anti h2bk123ub1

Yeast strains of the indicated genotypes (and their wild-type counterparts) were grown in YPD either at permissive or restrictive temperatures. Overnight-saturated cultures were diluted to an OD₆₀₀ of 0.2 and allowed to grow until they reached an OD₆₀₀ of 1. Five OD₆₀₀ equivalents of cells were lysed using a modified TCA extraction method as described (Keogh et al., 2006a (link), 2006b (link)). 10–20 mg of the lysates were separated by SDS-PAGE and variously probed with the following antibodies: anti-FLAG-M2 [for FLAG tagged Spt6] (Sigma-Aldrich, F1804; 1:5000), anti-G6PDH (Sigma-Aldrich, A9521; 1:100,000), anti-histone H3K4me3 (EpiCypher, 13–0004; 1:5000), anti-histone H3K79me3 (Abcam, ab2651, 1:2500) anti-histone H3K36me3 (Abcam, ab9050, ab9050; 1:1000), anti-histone H3 (EpiCypher, 13–0001; 1:50,000), anti-Spt16 (gift from Tim Formosa University of Utah, 1:5000), anti-H3K36me2 (Active Motif, 39255; 1:1000), anti-Set2 (Generated in the Strahl lab, 1:5000), anti-RNAPII-Ser2P (Active Motif, Clone #3E10, 61084; 1:100), anti-H2BK123ub1 (Cell Signaling Technology, 5546; 1:2000), and anti-H2B (Active Motif, 39237; 1:2000). HRP-conjugated anti-rabbit (GE Healthcare, NA934V; 1:10,000) and anti-mouse secondary (GE Healthcare, NA931V; 1:10,000), antibodies were used at 1:1000 and proteins were detected using ECL Prime or enhanced chemiluminescence ECL (Amersham Biosciences).