Figure 8.

Set4 is constitutively expressed in an erg3Δ strain under aerobic conditions by a precursor sterol and Upc2. (A) Modified pathway showing the final steps in ergosterol biosynthesis. The * on ERG3 represents potential pathways that modify episterol to generate additional sterol precursors (see Figure S5 in File S5). (B) qRT-PCR analysis of SET4 transcript levels in ergosterol mutant strains under aerobic conditions. Transcript levels were normalized to ACT1 and set relative to WT. (C) Western blot analysis of 3×FLAG-Set4 levels in WT and erg3∆ strain under aerobic conditions. The * indicates a likely protein degradation band. (D) Gene expression analysis by qRT-PCR of UPC2 in indicated strains under aerobic conditions. (E) SET4 mRNA levels determined by qRT-PCR in WT, erg3∆, upc2∆, and erg3∆upc2∆ under aerobic conditions. (B, D, E) * P < 0.05, ** P < 0.005, *** P < 0.0005, **** P < 0.0001. (F) Western blot analysis of Set4 protein levels in strains from E under aerobic conditions. G6PDH was used as a loading control and an untagged WT was used a negative control. The band under the Set4 band likely indicates protein degradation and is indicated by *. Gene expression data were normalized to ACT1 and set relative to WT. Error bars represents SD of three biological replicates. Gene expression and Western blot analyses were performed using the BY4741 strain.