Fig. 1
Functional relationship between Sgf73p and Yra1p. a Spotting assays to assess genetic interactions between yra1-1 and SAGA subunit-deletion mutants. Cells were spotted onto YPD plates with five-fold serial dilutions and incubated at 30 and 37 °C. b Spotting assays to assess the effects of Sgf73p overexpression in the yra1-1 mutant. Cells transformed with PGAL SGF73 inserted into the URA3 locus were cultured in YP + 2% raffinose (YPR) medium and plated on YPR and YPG (YP + 2% galactose) media. Plates were incubated at 30 and 37 °C. c In vitro pulldown assay of gel-filtration-purified Sgf73-DUBm against GST-tagged Yra1p. Binding was assessed by Western blotting against Sgf73p (α-HA) and Sus1p (α-FLAG). d In vitro pulldown assay of recombinant Sgf73p against GST-tagged Yra1p. Binding was assessed by Western blotting against Sgf73p (α-MBP). e In vitro pulldown assays were performed using Sgf73p (α-MBP) and the indicated subfragments of Yra1p (α-GST). f Co-immunoprecipitation (co-IP) assays of Sgf73p (α-13xMYC) and Yra1p (α-5xFLAG) in wildtype and rpt2-1 cells. Yra1p (α-5xFLAG) was immobilized on anti-FLAG M2 affinity gel and co-IP was assessed by Western blotting. g Co-IP assay of Ubp8p (α-13xMYC) and Yra1p (α-5xFLAG) in wildtype and sgf73Δ cells. Yra1p (α-5xFLAG) was immobilized on anti-FLAG M2 affinity gel and co-IP was assessed by Western blotting. h (Left panel) FISH analyses of YRA1, yra1-1, YRA1 sgf73Δ, and yra1-1 sgf73Δ. Cells were cultured at 30 °C to early log-phase and shifted to 37 °C for 2 h. Poly-(A) + RNA was detected using Cy3-labeled oligo(dT) probes and DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm. (Right panel) Quantification graph of poly-(A) + RNA intensity. Poly-(A) + RNA intensity was quantified using the same scan width of respective nuclei (determined by DAPI staining). WCE, whole-cell extract. Coomassie blue staining was used as a loading control
