Fig. 4: The Ume6 C-terminal region is essential for protein–protein interaction.

a, Diagram depicting Ume6 mutants. The green boxes indicate zinc-cluster DNA binding domains (DBDs), the blue boxes indicate 3× HA tag and the red line indicates an amino acid substitution (arginine to alanine). b, For biofilm formation, cells were grown in RPMI + 10% FBS at 37 °C for 24 h, and biofilms were stained with calcofluor-white. The side-view projection images represent three independent experiments (n = 3). c, Bar graph indicating biofilm volume measurements for each strain. Biofilm volume measurements were performed with three biological independent samples using ImageJ (n = 3). The values are the mean ± s.d. of three biological replicates. Statistical significance was determined using one-way ANOVA (Dunnett’s multiple-comparison test). Three biological replicates were used. d, Cells were grown in RPMI + 10% FBS at 37 °C for 4 h, and protein lysates from strains expressing the indicated HA- and/or FLAG-tagged protein were subjected to immunoprecipitation with anti-HA antibody. The precipitated proteins were detected using either anti-HA or anti-FLAG for western blot analysis. The cell lysates used for co-IP were used as an input control. The asterisks indicate non-specific bands. The images represent three independent experiments (n = 3). e, For biofilm formation, cells were grown in RPMI + 10% FBS at 37 °C for 24 h, and biofilms were stained with calcofluor-white. The side-view projection images represent three independent experiments (n = 3). f, Bar graph indicating biofilm volume measurements for each strain. Biofilm volume measurements were performed with three biological independent samples using ImageJ (n = 3). Values are the mean ± s.d. of three biological replicates. Statistical significance was determined using one-way ANOVA (Dunnett’s multiple-comparison test). g, ChIP-qPCR was performed using IP and input DNA from the WT (untagged), Ume6-HA and Ume6(R772A)-HA strains grown in RPMI + 10% FBS at 37 °C for 4 h. Values are the mean ± s.d. of three biological replicates. Statistical significance was determined using two-sided unpaired Student’s t-test. The images represent three independent experiments (n = 3). h, Cells were grown in RPMI + 10% FBS at 37 °C for 4 h, and protein lysates from strains expressing the indicated HA- and/or FLAG-tagged protein were subjected to immunoprecipitation with anti-HA antibody. The precipitated proteins were detected using either anti-HA or anti-FLAG for western blot analysis. The cell lysates used for co-IP were used as input control. The asterisk indicates a non-specific band. The images represent three independent experiments (n = 3). P < 0.0001; the exact P values were <0.0001 for all indicated comparisons. All strains used in this figure are homozygous for alleles indicated. The genotype of the untagged strain is UME6^+/+.