FIG 8.
SDS-PAGE and phosphatase analysis of the mobility shift of hyperactive Tac1. (A) Immunoblot of Tac1 SDS-PAGE mobility in lysates from a 6His3Flag-tagged wild-type Tac1 strain (yLM485) treated with 10 μg/ml FNZ, 10 μg/ml EST, or 40 μg/ml fluconazole (FLC) for the indicated periods of time. Samples were resolved on 6% SDS-PAGE gel and the blot was probed with an anti-Flag antibody. Coomassie blue staining (CBS) was used as the loading control. (B) Immunoblot analysis of SDS-PAGE mobility in lysates from strains expressing an endogenous level of individual 6His3Flag-tagged Tac1 variants (wild-type [yLM485], N972D [yLM534], Δ962–969 [yLM535], ΔM677 [yLM533], R693K [yLM532], and A736V [yLM531]) in the absence and presence of 10 μg/ml fluphenazine. Samples were resolved on 6% SDS-PAGE gel and the blot was probed with an anti-Flag antibody. CBS was used as the loading control. (C) Immunoblot analysis demonstrating the effect of λ protein phosphatase (λ-pp) treatment on the SDS-PAGE mobility shift of hyperactive Tac1 purified from strains that overexpressed 6His3Flag (HF)-tagged wild-type Tac1 (yLM530; grown in the presence and absence of fluphenazine), Tac1R693K (yLM540), or Tac1N977D (yLM536). Samples were resolved by 6% SDS-PAGE and the blot was probed by an anti-Flag antibody. (D) Immunoblot analysis of the effect of λ protein phosphatase treatment (in the presence and absence of 50 mM NaF and 20 mM Na3VO4 [F−/VO43−] phosphatase inhibitors) on the SDS-PAGE mobility shift of Tac1 purified from 6His3Flag-tagged wild-type Tac1 cells (yLM530) grown in the absence and presence of fluphenazine. Samples were resolved by 6% SDS-PAGE and the blot was probed by an anti-Flag antibody.