galactose inducible pyes2 vector  (Vector Laboratories)

Journal: PNAS Nexus

Article Title: Ancient and animal-specific regulatory modes of EWS::FLI1 revealed by a minimal yeast model

doi: 10.1093/pnasnexus/pgag329

Figure Lengend Snippet: Transcriptional activation by EWS::FLI1 through recognition of GGAA microsatellites in yeast. A) W303-1A derivatives carrying pCYC1 (top), 20 × (GGAA) (middle), or 40 × (GGAA) (bottom) reporters transformed with empty pYES2, EWS::FLI1, or EWS::FLI1 RRLL . Left, representative confocal images (bright-field, top; GFP fluorescence, bottom); middle, flow-cytometry histograms of cell counts as a function of Comp-GFP; right, mean GFP fluorescence. B) Time-course of 20×(GGAA)_ pCYC1 _eGFP cells transformed with pYES2 carrying EWS::FLI1 RRLL (top) or EWS::FLI1 (bottom). Samples collected at the indicated times were immunoblotted with anti-EWSR1 and anti-GFP monoclonal antibodies; arrowheads indicate EWS::FLI1 and GFP. Ponceau staining is shown as a loading and transfer control. Equivalent results were obtained in two further biological replicates. C) Relative EWS::FLI1 and GFP levels, normalized to the mean EWS::FLI1 signal at 6 h of induction (100%). Error bars, SD. * P < 0.05; ns, not significant; two-way ANOVA.

Article Snippet: EWS::FLI1 and its DNA-binding (EWS::FLI1 RRLL ) and prion-like-domain (EWS::FLI1 YS37 ) mutants were expressed from the galactose-inducible pYES2 vector ( URA3 ) in S. cerevisiae W303-1A.

Techniques: Activation Assay, Yeast, Transformation Assay, Confocal, Fluorescence, Flow Cytometry, Bioprocessing, Staining, Control

Journal: The EMBO Journal

Article Title: Multifunctional roles of Brl1-Brr6 in nuclear envelope fusion during nuclear pore complex biogenesis

doi: 10.1038/s44318-026-00718-y

Figure Lengend Snippet: ( A ) AαH predictions for the wild-type and mutant sequences of Brr6 were generated using HeliQuest (Gautier et al, ). The upper cartoon illustrates the domain organization of Brr6, highlighting the two transmembrane (TM) domains, the AαH, and the four conserved cysteine residues. Amino acid positions are indicated. ( B ) Subcellular localization of GFP-tagged Brr6 wild-type and brr6 L145E AαH region. The pGal1 promoter was expressed for 3 h by the addition of galactose. Fluorescence microscopy was performed to assess localization. dsRed-HDEL was used as NE and ER marker (Madrid et al, ). Size bar: 5 µm. Representative image from three independent repeats. ( C ) Growth assay of the BRR6 shuffle strain carrying the empty LEU2 -based plasmid pRS315 or pRS315 containing the indicated BRR6 alleles ( BRR6 , brr6 L145E , and brr6 F152E ). Tenfold serial dilutions were spotted onto SC–LEU and 5-FOA plates and incubated at 30 °C for 2 days. Growth on 5-FOA plates selects for cells that have lost the URA3 plasmid, thereby testing whether the LEU2 -based plasmid alone can support viability in the absence of wild-type BRR6 . One representative of three independent experiments is shown. ( D ) Wild-type yeast cells carrying the indicated pGal1 plasmids were spotted in tenfold serial dilutions onto glucose (Glu) or galactose/raffinose (Gal/Raf) plates and incubated at 30 °C. One representative of three independent experiments is shown.

Article Snippet: Cells overexpressing these mutant alleles displayed severe growth defects on the inducing galactose plates (Gal/Raf) while control cells (pGal1- BRR6 and pGal1 vector) showed robust growth (Fig. ).

Techniques: Mutagenesis, Generated, Fluorescence, Microscopy, Marker, Growth Assay, Plasmid Preparation, Incubation