Fig. 2

Participation of Spt7 in Ubp8-dependent H2B deubiquitination. a Whole cell extracts were obtained from WT, sgf73Δ and spt7Δ cells, and levels of H2Bub1 were monitored by western blotting using an anti-α-H2Bub1 antibody. Levels of Pgk1 and total H2B were analyzed as loading controls. Cropped blots are shown for clarity. Full-length blots are presented in Additional file 2: Fig. S4 Rep1/2/3 shown in the figure correspond to three independent replicas. b Quantification of panel a showing the ratio of H2Bub1/H2B of three independent experiments. Error bars represent standard deviation. c Ubp8-TAP was purified via TAP from wild-type (WT, lane 2), sus1Δ (lane 3) and spt7Δ (lane 4) strains. The purified Ubp8-TAPs were then incubated with purified histone H2B (containing HA-ubiquitin modified H2B and unmodified H2B) and in vitro H2Bub1 deubiquitination was assayed. Purified histone H2B alone was incubated with buffer and used as a negative control (lane 1(−)). H2B monoubiquitin levels are indicated at the bottom relative to the level in lane 1(−), which was given an arbitrary value of 1. The values are representative of at least three independent experiments. Cropped blots are shown for clarity. Full-length blots are presented in Additional file 2: Fig. S5