Figure 2.

The expression level of Adh1 in the glucose-grown Δssb1Δssb2 strain is promoter-dependent. (A) Schematic representation of chromosomal ADH1 under the control of its endogenous regulatory elements and plasmid encoded Flag-tagged ADH1 under the control of the ZUO1 regulatory elements. The N-terminal Flag tag is indicated in gray. (B) Aliquots of total protein extracts of wild-type and Δssb1Δssb2 cells prepared as described in Figure 1A were analyzed via immunoblotting with antibodies recognizing Adh1, the Flag tag, or Zuo1 as indicated. Flag-tagged Adh1 is recognized by the Adh1 antibody as well as by the Flag tag antibody. The Flag-tagged version can be separated from the untagged version on 10% TRIS-Tricine gels and is not shown on the blot developed with the Adh1 antibody. Endogenous Zuo1 was monitored as a control. (C) Quantification of the relative amounts of Adh1 and Flag-Adh1 in wild type and Δssb1Δssb2. Densitometric analysis of immunoblots was performed in the linear range as described in Experimental Procedures. The expression level of endogenous and Flag-tagged Adh1 was determined in three independent clones. Expression in wild type (white bars) was set to 1. (Hatched bars) Expression level in Δssb1Δssb2. Error bars represent the standard error of the mean.