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. Author manuscript; available in PMC: 2012 Aug 5.
Published in final edited form as: Mol Cell. 2011 Aug 5;43(3):381–391. doi: 10.1016/j.molcel.2011.05.033

Figure 2. WDR5 is ubiquitinated by CRL4B in vivo and in vitro.

Figure 2

(A) HCT116 cells transfected with indicated plasmids were lysed and subjected to immunoprecipitation (IP) with antibody to WDR5 under denaturing condition. The resulting precipitates were subjected to western analysis (IB) with antibodies to HA or to WDR5. A portion of the input lysates was also subjected directly to immunoblot analysis with antibodies to myc or tubulin.

(B) HCT116 cells transfected with plasmids for HA-ubiquitin along with siRNA targeting DDB1, CUL4A or CUL4B were lysed and subjected to immunoprecipitation (IP) and immunoblot analysis (IB) as in (A).

(C) Recombinant WDR5 was incubated with different combinations of E1, E2 and CRL4B (E3) along with ubiquitin and ATP. Reactants were subjected to immunoblot analysis (IB) with indicated antibodies.

(D) Lysates of HEK293T cells treated with MG132 for 6 hours were subjected to immunoprecipitation with indicated antibodies and the resulting precipitates, as well as a portion (10% of the input for immunoprecipitation) of the cell lysates, were subjected to immunoblot analysis (IB) with indicated antibodies.

(E) HEK293T cells were transiently transfected with indicated vectors, treated with MG132 for 6 hours and harvested. Protein bindings were determined by IP-western analysis using indicated antibodies.