Fig. 2.
Enrichment of ubiquitylated RNAPII species. (A) A sketch illustrating the principle of Dsk2 pulldown strategy (the same principles apply to MultiDsk). Dsk2 binds the ubiquitin moiety on proteins and this property is exploited to enrich ubiquitylated proteins from the extract. Note that, if used as indicated in text, this method is capable of depleting the extract of ubiquitylated proteins. (B) Kinetics of RNAPII ubiquitylation upon UV irradiation in human cells. HEK293 TRex flpIn cells were irradiated with 20 J/m2 UVC, and samples were taken at different timepoints after irradiation. Dsk2 analysis was performed as described in text. Note that different exposures of the same Western blot membranes are shown. Approximate positions of the protein markers are indicated on the left. mUb, mono-ubiquitylated RPB1. pUb, poly-ubiquitylated RPB1. “Ub?”, slower migrating RPB1 band, which may represent ubiquitylated species in the extract. (C) Analysis of RNAPII ubiquitylation upon 4-NQO treatment in HEK293 TRex flpIn cells. The cells were treated with the indicated doses of 4-NQO for 1 h, and Dsk2 analysis was performed as described in the text.