Figure 3
Drug resistance levels (MIC FLC ) of Cdr1p-expressing strains are directly proportional to the amount of Cdr1p expressed. A. SDS-PAGE of plasma membrane proteins (30 μg) isolated from AD∆ strains containing different Sfi I stem-loop constructs. The black arrowhead indicates Cdr1p and the white arrowhead indicates the prominent plasma membrane proton pump protein Pma1p. wt = AD∆/P-CDR1-URA3; SfiI = AD∆/construct9-CDR1; lanes labeled 8, 2 and 0 represent Cdr1p expressing strains with decreasing loop-size of 8 nucleotides (AD∆/construct6-CDR1), 2 nucleotides (AD∆/construct7-CDR1) or no loop at all (AD∆/construct8-CDR1); lanes labeled 5, 6 and 7 represent strains with increasing stem-size of 5 GC-pairs (AD∆/construct11-CDR1), 6 GC-pairs (AD∆/construct14-CDR1) and 7 GC-pairs (AD∆/construct15-CDR1). B. The MICFLC values for each construct correlated well with the amounts of Cdr1p expressed (measured as pixels using the ImageJ software [30]). %CDR1 (Y-axis) and %MICFLC (X-axis) are the expression levels and MICFLC relative to wt Cdr1p. To the right is a graphical illustration of this correlation (constructs #14 and #15 were excluded from the graph because their Cdr1p expression was below the detection limit but the MICFLC = 0.5 of the negative control strain AD (no Cdr1p) was included), and the dashed grey line shows the theoretical trend line expected for a direct linear correlation between MICFLC values and the amounts of Cdr1p expressed.