Extended Data Fig. 8: Targeted degradation of RPL20B in yeast leads to altered mRNA expression of cytokines.
From: Profiling phagosome proteins identifies PD-L1 as a fungal-binding receptor
a, Schematic of auxin-induced degradation of RPL20B in the engineered mAID-RPL20B S. cerevisiae strain. b, The CRISPR-cas9 genome editing strategy for introducing the mAID and HA tag to the S. cerevisiae Rpl20b locus. The sequences of the guide RNA (gRNA), or the mutated gRNA in the edited genome are underlined. Silent mutation or insertion introduced are shown in red. c, Immunoblotting of RPL20B in ribosomes purified from the indicated yeast. d, ELISA assay determining binding of hPD-L1-Fc to ribosomes purified from the indicated yeast with technical triplicates. e-g, BMDMs were stimulated or not with RPL20B-low or control yeast for 4 h and mRNA expression was assessed by RNA-seq. e, Venn diagram showing the number and percentage of differentially expressed genes by the indicated comparisons. Adjusted P value cutoff, 0.05. f, Pathway enrichment bubble plot showing the most significantly enriched pathways by comparing the transcriptomes of BMDMs treated with RPL20B-low or control yeast. g, Heatmap showing expression of combined leading-edge genes from the top 2 enriched pathways in (f). NES, normalized enrichment score. Data are mean + S.E.M. (d). Data are representative of 2 independent experiments (c, d). P values by one-way ANOVA with Tukey’s multiple comparisons tests (d). ****P < 0.0001.
