Journal: bioRxiv

Article Title: Disruption of the nascent polypeptide-associated complex leads to reduced polyglutamine aggregation and toxicity

doi: 10.1101/2024.04.19.590245

Figure Lengend Snippet: (A) Yeast 74-D694 WT and NAC deletion strains expressing Gal1-inducible EV, htt25Q, or htt103Q constructs were serially diluted 5-fold and spotted onto ¼ YPD (not shown), SD-ura, and S-ura, 2% galactose (first 3 spots shown) to monitor expanded polyglutamine toxicity and the ability of nac deletion to rescue expanded polyglutamine cytotoxicity (n=3). (B) A yeast 74-D694 WT strain expressing EV, EGD2, or EGD1 constructs in combination with Gal-inducible EV or htt103Q constructs were serially diluted 5-fold on ¼ YPD (not shown), SD-ura-his, and S-ura-his, 2% galactose, 0.1% raffinose (first three spots shown) to evaluate expanded polyglutamine toxicity and the ability of EGD1 and EGD2 overexpression to rescue expanded polyglutamine cytotoxicity (n=3).

Article Snippet: Plasmids expressing alpha-synuclein constructs controlled by the inducible GAL1 promoter (pRS426-Gal1-SNCA WT -GFP, pRS426-Gal1-SNCA A30P -GFP, pRS426-Gal1-SNCA A53T -GFP) were kindly gifted by M. Jackrel. pTH726-CEN-RLuc/minCFLuc and pTH727-CEN-RLuc/staCFLuc were a gift from Tobias von der Haar (Addgene plasmid # 38210 and 38211).

Techniques: Expressing, Construct, Over Expression

Journal: bioRxiv

Article Title: Disruption of the nascent polypeptide-associated complex leads to reduced polyglutamine aggregation and toxicity

doi: 10.1101/2024.04.19.590245

Figure Lengend Snippet: (A) WT and NAC deletion strains expressing Gal1-htt25Q-CFP or Gal1-htt103Q-CFP constructs were grown in selective media in the presence of 2% galactose for 6 and 20 hours. Two-dimensional z-stack images were taken with a Zeiss LSM 880 Airyscan Two-Photon Confocal Microscope with a 63x oil immersion objective. The egd1Δbtt1Δ strain showed a smaller population of cells with aggregates than the WT or egd1Δegd2Δ strains at both time points. (B) Quantification of percent cells expressing Gal1-htt25Q-CFP or Gal1-htt103Q-CFP constructs containing aggregates after 6 hours induction by 2% galactose in selective media. Significance was determined by Fisher’s exact test (****=<0.0001). (C) Quantification of percent cells expressing Gal1-htt25Q-CFP or Gal1-htt103Q-CFP constructs containing aggregates after 20 hours induction by 2% galactose in selective media. Significance was determined by Fisher’s exact test (****=p value<0.0001). (D) Representative images of cells with 0, 1, 2, or 3 aggregates. Microscopy of WT and NAC deletion strains expressing Gal1-htt25Q-CFP or Gal1-htt103Q-CFP constructs for 6 and 20 hours were evaluated based on this scale. (E) WT and NAC deletion strain cells expressing Gal1-htt103Q-CFP constructs for 6 or 20 hours and containing aggregates were evaluated as having 1, 2, or 3 or more aggregates. The resulting population distributions are represented as percentages in pie graphs.

Article Snippet: Plasmids expressing alpha-synuclein constructs controlled by the inducible GAL1 promoter (pRS426-Gal1-SNCA WT -GFP, pRS426-Gal1-SNCA A30P -GFP, pRS426-Gal1-SNCA A53T -GFP) were kindly gifted by M. Jackrel. pTH726-CEN-RLuc/minCFLuc and pTH727-CEN-RLuc/staCFLuc were a gift from Tobias von der Haar (Addgene plasmid # 38210 and 38211).

Techniques: Expressing, Construct, Microscopy

Journal: bioRxiv

Article Title: Disruption of the nascent polypeptide-associated complex leads to reduced polyglutamine aggregation and toxicity

doi: 10.1101/2024.04.19.590245

Figure Lengend Snippet: (A) WT and NAC deletion strains expressing Gal1-FLAG-htt25Q-CFP or Gal1-FLAG-htt103Q-CFP constructs were grown in selective media in the presence of 2% galactose for 6 hours prior to SDS-PAGE and Western blotting for FLAG and PGK1. Western blots are representative images of three independent experiments. (B) WT and NAC deletion strains expressing Gal1-FLAG-htt25Q-CFP (not shown) or Gal1-FLAG-htt103Q-CFP constructs were grown in selective media in the presence of 2% galactose for 6 hours prior to Filter Trap Assay and Western blotting for FLAG. Western blot is a representative image of three independent experiments. (C) Quantification and normalization to WT of 3 independent Filter Trap Assay Western blots represented in 4(B), data are represented as mean ± SEM, significance was determined by paired t-test, *=p value<0.05, and ns=p value>0.05. (D) Western blot image showing the Ubiquitinated proteins in WT and egd1Δbtt1Δ strains co-expressing htt25Q, htt103Q constructs with an Ub-X- LacZ reporter: pGal-Ub-P- LacZ . Pgk1 showing the loading control. Representative image shown here.

Article Snippet: Plasmids expressing alpha-synuclein constructs controlled by the inducible GAL1 promoter (pRS426-Gal1-SNCA WT -GFP, pRS426-Gal1-SNCA A30P -GFP, pRS426-Gal1-SNCA A53T -GFP) were kindly gifted by M. Jackrel. pTH726-CEN-RLuc/minCFLuc and pTH727-CEN-RLuc/staCFLuc were a gift from Tobias von der Haar (Addgene plasmid # 38210 and 38211).

Techniques: Expressing, Construct, SDS Page, Western Blot, TRAP Assay, Control