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. 2013 Oct 14;8(10):e76424. doi: 10.1371/journal.pone.0076424

Figure 1. Overexpression of IRC20 causes transcription phenotypes.

Figure 1

A) Yeast strain GY2205, which contains the suc2Δuas(−1900/−390) reporter and endogenous TAP-IRC20, was transformed with either empty vector (pRS425) or a 2 μ TAP-IRC20 plasmid (pAR41) and assayed for the Bur phenotype on a YPsucrose plate (top). A western blot to detect expression of TAP-Irc20 is shown below, along with G6PDH loading control. B) 2 μ GAL1pr-IRC20 exhibits an Spt- phenotype. Yeast strains GY482 (his4–912δ lys2–128δ) and GY2203 (his4–912δ lys2–128δ GAL1pr-TAP-IRC20) were transformed with the indicated plasmids and transformants were assessed for their Spt phenotype on SC-His and SC-Lys plates. C) Overexpression of epitope-tagged Irc20. Irc20 was HA-tagged at its N- or C-terminus and expressed from a 2 μ plasmid. The 2 μ Bur- phenotype was disrupted by the C-terminal 6HA tag (pAR9), while N-terminal 3xHA-Irc20 fusion (pAR11) remained functional. D) N-terminally HA-tagged D534A E535A irc20 ATPase (pAR30) and C1239A RING finger (pAR14) mutants were created and expressed from a 2 μ plasmid in yeast strain GY460. Both mutants are expressed at similar levels as wild-type Irc20, but are incapable of producing the 2 μ Bur- phenotype.