Hel1, Hel2, Pep5 and Snt2 interact with the E2 enzyme Ubc4 as well as histones.

<p>(<b>A</b>) <i>Hel1, Hel2, Pep5 and Snt2 interact with Ubc4</i>. Whole cell extracts prepared from 0.5 liter cultures of the indicated strains were used to IP Ubc4-MYC essentially as described for Asf1-FLAG IPs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036295#pone-0036295-g003" target="_blank">Figure 3</a>, but using anti-MYC EZ view beads (Sigma). Co-IPed HA-tagged E3 enzymes were detected using HA.11 antibodies. Apart from the predominant bands corresponding to the full length proteins (indicated by the asterisks), additional bands of weaker intensity are detected and these are likely to be either degradation products (faster migrating) or ubiquitylated (slower migrating) forms of these proteins. No co-IPed proteins were detected in the parental untagged strain carrying Ubc4-MYC but lacking HA-tagged proteins. 1% of the whole cell extracts were loaded on a separate gel to determine that roughly equal amounts of proteins were present in the input fraction by measuring the amount of histone H4. (<b>B</b>) <i>Hel1, Hel2, Pep5 and Snt2 interact with histones</i>. The HA-tagged E3 enzymes were IPed from 1 liter cultures of the indicated strains using HA.11 antibodies. Co-IPed endogenous histones H3 and H4 was detected using the polyclonal antibodies described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036295#pone.0036295-Gunjan1" target="_blank">[6]</a>. No co-IPed histones were detected in the “no tag” control. The amount of Ubc4-MYC was measured in 1% of the input fraction to demonstrate that roughly equal amounts of cell extracts were used for the IP reactions. (<b>C</b>) <i>Hel1 and Hel2 interact with Rad53</i>. MYC tagged Hel1 or Hel2 were IPed using MYC antibody beads from 3 liter cultures of strains carrying FLAG-tagged Rad53. IPed and Co-IPed proteins were detected by Western Blotting using appropriate antibodies. The relative amount of histone H3 present in each of the “input” extracts used for the IP is shown to demonstrate that same amount of material was used for each IP. (<b>D</b>) <i>Snt2 interacts with Rad53 whereas Pep5 does not</i>. Tandem Affinity Purification (TAP) tagged Pep5 or Snt2 was IPed from 3 liter cultures of strains carrying FLAG-tagged Rad53. Any co-IPed Rad53 was detected using FLAG antibodies. The similar levels of Rad53-FLAG present in the extracts confirm that the same amount of extracts was used for each IP. No evidence for any interaction between Rad53 and Pep5 was observed even upon scaling up the Pep5-TAP IP by 3-fold (data not shown).