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. Author manuscript; available in PMC: 2011 Jul 7.
Published in final edited form as: Mol Cell. 2011 Jan 7;41(1):82–92. doi: 10.1016/j.molcel.2010.12.017

Figure 4. Rpb1 accumulates in the ubiquitinated state in cdc48-3, ubx4Δ, ubx5Δ and ubx4Δubx5Δ mutants.

Figure 4

(A) WT (W303 and 204) and mutant cells were shifted to 37°C for 90 minutes and UV irradiated. Lysates were fractionated on a GstDsk2 resin. The input extract and bound fractions were immunoblotted (IB) for Rpb1 (4H8) and Ub, as indicated. 204 is RJD487, a wild-type congenic to cdc43-2, and untreated sample and beads were partially lost during IP.

(B) Rpb1 Ub conjugates accumulated in ubx5Δ but not ubx1Δ, and were diminished in cul3Δ mutant cells. Methods same as (A) except that cells were maintained at 30°C. Asterisk indicates sumoylated Rpb1.

(C) Rpb1 Ub conjugates hyperaccumulated in a ubx4Δubx5Δ double mutan. Methods same as (A) except that cells were maintained at 30°C.

(D) Ub conjugates accumulated on proteasomes isolated from ubx4Δ and ubx5Δ mutants. Lysates from wildtype and mutant cells expressing Flag-tagged Pre1 (or not) were immunoprecipitated (IP) with anti-Flag and immunoblotted (IB) for Ub, Rpn11 and 20S. Input extracts were blotted for 20S.

See also Figure S5.