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. 2022 Oct 25;41(4):111536. doi: 10.1016/j.celrep.2022.111536

Figure 3.

Figure 3

Smy2 works with Cdc48 and affects protein ubiquitylation

(A) Dilution series of cdc48-3 cells (carrying the indicated 2-μm plasmids) plated on minimal media and grown at the specified temperatures for 2–4 days.

(B) Dilution series as in (A), showing the effect of cdc48 mutation, deletion of SMY2, or both.

(C) Western blot of ubiquitylated proteins from wild-type (WT) and smy2Δ cells before or after DNA damage with 4-NQO.

(D) As in (A) but ubiquitylated proteins isolated by MultiDsk affinity chromatography. Here, a 3%–8% gel was run until the 75-kDa band had reached the bottom of the gel. The Ponceau S stain (bottom panel) shows the eluted GST-MultiDsk protein.

(E) Top, western blot showing coIP of RNAPII (Rpb1) with FLAG-tagged Smy2 at different times after DNA damage. Bottom, inputs. Note that it was not possible in these experiments to detect poly-ubiquitylated Rpb1 due to the weak signal.