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. Author manuscript; available in PMC: 2012 Feb 18.
Published in final edited form as: Mol Cell. 2011 Feb 18;41(4):384–397. doi: 10.1016/j.molcel.2011.01.024

Figure 3. WAC regulates H2B ubiquitination.

Figure 3

(A) Upper panel: 293T cell lysates were analyzed by IP and western blotting with the indicated antibodies. Lower panel: 293T cell expressing wild type or mutant WAC were analyzed by IP and western blotting with the antibodies indicated. The expression level of WAC and its mutants were examined by anti-FLAG antibody. (B–D) HCT116 cells were treated with indicated siRNA. Cell lysates were IPed and blotted with the indicated antibody. The level of WAC, RNF20 and hRAD6 was blotted from whole cell lysates. (B Right panel): The input level of RNF20 was normalized, the interaction between RNF20 and hRAD6 was examined. (E) HCT116 cells were treated with the indicated siRNA. Histone marks were examined with the indicated antibodies. Blots with anti-actin and anti-H3 were used as protein loading controls. (F) Coomassie blue staining of purified protein. (G) WAC facilitates RNF20/40 and hRAD6-mediated H2B ubiquitination in vitro. In vitro chromatin ubiquitination assays contained nucleosomal histones, hE1, hRAD6A, RNF20/40, HA-tagged ubiquitin, and GST-WAC (0, 50, 0, 10, 25, 50 ng from lane 1 to 6). Nucleosomal histones were examined by western blotting with anti-ubH2B antibody (upper panel) and then anti-H2B antibody (lower panel).