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. 2017 Oct 24;61(11):e01342-17. doi: 10.1128/AAC.01342-17

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FIG 9 — Effect of SSN3 and MED3 on the hyperactive Tac1 SDS-PAGE mobility shift. (A) ChIP analysis of Ssn3 occupancy at the CDR1_DRE in strains with either two copies of native SSN3 (WT/WT; BWP17) or C-terminal 3×HA-tagged SSN3 (HA/HA; yLM481), grown in the presence and absence of fluphenazine, using an anti-HA antibody. Recovery rate (% Input) of DNA fragments containing the 1-DRE amplicon in uninduced BWP17 was set to 1 to calculate the relative recovery at additional regions. (B) Immunoblot analysis of the Tac1 SDS-PAGE phosphorylation shift in lysates from a 6His3Flag-tagged Tac1 SSN3^+/+ strain (yLM485) and its ssn3Δ/Δ derivative (yLM552) grown in the absence and presence of fluphenazine. For all immunoblots in the figure, samples were resolved by 6% SDS-PAGE and probed by an anti-Flag antibody, and Coomassie blue staining (CBS) was used as the loading control. (C) Immunoblot analysis of the fluphenazine-induced Tac1 SDS-PAGE phosphorylation shift in lysates from strains (yLM596 and yLM660) that overexpressed 6His3Flag-tagged wild-type Tac1 in an ssn3Δ/Δ strain complemented by either wild-type SSN3 (yLM279) or a kinase-dead allele, SSN3^D325A (yLM276). (D) Immunoblot analysis of the SDS-PAGE phosphorylation shift of Tac1^GOF mutant proteins in lysates from strains expressing individual 6His3Flag (HF)-tagged Tac1^GOF mutant variant (Tac1^R693K [yLM532], Tac1^Δ962–969 [yLM535], or Tac1^N972D [yLM534]), their ssn3Δ/Δ derivatives (yLM553, yLM554, and yLM556, respectively), and their med3Δ/Δ derivatives (yLM550, yLM551, and yLM549, respectively). Cell lysates generated from a 6His3Flag-Tac1^WT-expressing strain (yLM485) before and after fluphenazine treatment are included for comparison. (E) Immunoblot analysis of the Tac1 SDS-PAGE phosphorylation shift in lysates from a 6His3Flag-tagged Tac1 MED3^+/+ strain (yLM485) and its med3Δ/Δ derivative (yLM547) grown in the presence of fluphenazine for the indicated period of time. (F) Immunoblot analysis of the Tac1 SDS-PAGE phosphorylation shift in lysates from strains expressing 6His3Flag-tagged wild-type Tac1 (yLM485) or TAD truncated Tac1^1–845 (yLM539) treated with fluphenazine for the indicated periods of time.