Journal: Cell Death & Disease
Article Title: Arginyltransferase1 drives a mitochondria-dependent program to induce cell death
doi: 10.1038/s41419-025-07917-1
Figure Lengend Snippet: A Left panel displays representative Western blots depicting global ubiquitination levels in ate1 Δ yeast with either pGAL: ATE1-GFP or empty vector, which were induced for 6 h with 2% galactose in liquid media. The level of Ate1-GFP was probed with anti-GFP. Pgk1 was used as a loading control. The right panel display quantification of the fold-change of total ubiquitin ladder signals by switching from glucose to galactose media (with raffinose media as a transition condition), which was normalized by Pgk1 loading. A p value > 0.05 is considered nonsignificant “ n.s .” ( n = 4). B Representative Western blot showing the expression levels of cytosolic stress response protein HSP70 in the yeast cells carrying the pYES2-pGAL: ATE1-6 × His- URA3 expression vector or the empty control vector, which were induced with 2% galactose for 6 h in liquid media. The level of Ate1 was probed with antibody against 6 × His tag. The lower panel shows the densitometric analysis of the cytosolic Hsp70 levels as expressed in fold difference after normalization to the internal protein loading control Pgk1 ( n = 6). C Similar to ( B ), except that the level of Grp78/HDEL, a maker of the endoplasmic reticulum unfolded protein stress response, was shown. The quantification was based on 3 independent repeats ( n = 3). D Growth assay of WT or ump1 Δ yeast carrying either pYES2-pGAL: ATE1-GFP- URA3 or empty vector. The growth was measured by a serial dilution growth assay on Ura-minus SD plates containing 2% glucose or 2% galactose, where the expression of Ate1 is non-induced or induced, respectively. Plates were incubated at 30 °C and images were taken after 3 days. E Similar to ( D ), except that ubr1 Δ was used to compare to WT yeasts.
Article Snippet: Western blot blocking reagent was obtained from Roche (catalogue number 75255200) The primary antibodies include: monoclonal mouse anti-GFP (from Roche, clone 7.1 and 13.1, Cat# 11814460001) Rabbit anti-yeast alpha tubulin (Abcam EPR13799 ) anti-yeast- phosphoglycerate kinase1 (Pgk1) (Thermofischer scientific # 459250), Rabbit anti-yeast Cmc2 was a gift from Dr. Antonio Barrientos (University of Miami) mouse anti-Grp78 (SCBT# HDEL Antibody (2E7): sc-53472) HSP70 Monoclonal antibody (Proteintech catalogue# 66183-1) The custom-produced rabbit anti-RDD antibody was ordered from Genscript INC as described in our previous work [ ].
Techniques: Western Blot, Ubiquitin Proteomics, Plasmid Preparation, Control, Expressing, Growth Assay, Serial Dilution, Incubation
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