Fig. 5
Sgf73p is required for heat shock survival and proteostasis. a (Upper panel) De novo motif enrichment analysis of Sgf73p-binding sites was performed using HOMER. (Lower panel) Comparison of the Sgf73p-binding motif with known motifs showed significant correspondence with the Hsf1p-binding motif. b Heatmaps of ChIP-seq signals representing Sgf73p-binding (α-FLAG) in wildtype, rpt2-1, and spt20Δ cells, from 1 kb upstream to 0.5 kb downstream of the TSS regarding Hsf1-dependent genes (n = 18) before and after heat shock. Heatmaps were sorted in descending order of Sgf73p enrichment. The y-axis color scale indicates normalized ChIP-seq read count. c ChIP-qPCR analysis against Sgf73p (α-FLAG) of the GAL1 gene before and after galactose induction in mock, wildtype, and rpt2-1 cells. d Cell survival analysis after heat shock was assessed by spotting assays. Wildtype, rpt2-1, and sgf73Δ cells were subjected to heat shock at 52 °C for 15 min, plated on YPD medium in five-fold serial dilutions, and incubated at 25 °C for 3 days. e Visualization of GFP-Ubc9ts by fluorescence imaging. Wildtype, rpt2-1 and sgf73Δ cells carrying the galactose-inducible GFP-Ubc9ts construct were subjected to heat shock at 37 °C for 2 h, and then fixed and imaged. Scale bar, 5 μm. f GFP-Ubcp9ts protein levels (α-GFP) in wildtype, rpt2-1, and sgf73Δ cells subjected to heat shock were assessed by Western blotting. β-Actin was used as a loading control. Standard deviations of three independent experiments are shown by error bars. kb, kilobase. TSS, transcription start site
