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. 2021 Apr 26;11(7):jkab134. doi: 10.1093/g3journal/jkab134

Figure 3.

Figure 3

sch9Δ and sns1 mutations have opposing effects on the expression of CAT8, ADR1, and HAP4. (A) β-galactosidase activities of CAT8-lacZ, ADR1-lacZ, and HAP4-lacZ reporter genes in the BY4741 wild type strain grown in YNBcas5D (Dextrose) and YNBcasR (Raffinose) medium. β-galactosidase activity assays were carried out as described in Materials and Methods. (B) β-galactosidase activities of CAT8-lacZ, ADR1-lacZ, and HAP4-lacZ reporter genes in haploid wild type (WT, BY4741), sch9Δ (PPY632), sns1 (PPY730), and sch9Δ sns1 (PPY566) mutant strains and corresponding diploid strains (WT × WT, PPY745, PPY737, PPY741) grown in YNBcas5D and YNBcasR medium. The reporter gene activity in wild type haploid and diploid strains grown in dextrose and raffinose medium was set as 1, respectively. (C) β-galactosidase activities of CAT8-lacZ, ADR1-lacZ, and HAP4-lacZ reporter genes in haploid wild type (WT, BY4741), sns2 (ZLY6614), and sch9Δ sns2 (ZLY6619) mutant strains grown in YNBcas5D and YNBcasR medium. (D) β-galactosidase activities of CAT8-lacZ, ADR1-lacZ, and HAP4-lacZ in the W303 background strains. Wild type haploid (W303-1B), sch9Δ mutant haploid (PPY622), wild type diploid (W303-1A × W303-1B), and sch9Δ homozygous mutant diploid cells (PPY622 × PPY624) were grown in YNBcas5D or YNBcasR. (E and F) Western blots of Cat8-HA and Hap4-HA in wild type and mutant diploid strains as indicated. Wild type (BY4741 × BY4742), sch9Δ × sch9Δ (PPY745), sns1 × sns1 (PPY737), and sch9Δ sns1 × sch9Δ sns1 (PPY741) mutant strains carrying a plasmid encoding CAT8-HA (pZL3929) (panel E) or HAP4-HA (pZL1324) (panel F) were grown in YNBcas5D and YNBcasR medium. The left and right panels of Cat8-HA were from the same membrane, but the Western blot from dextrose-grown cells required a longer exposure time due to weak signals. The arrowhead indicates the position of Cat8-HA bands. Ponceau S staining was used as the loading control. (G and H) Quantification of the ratios of Cat8-HA (panel G) and Hap4-HA (panel H) protein amount to the loading control by Ponceau S staining. The ratio of HA-tagged protein/loading control was set as 1 in wild type cells. The data are presented as the mean ± standard deviation, n = 2.