fluorogenic proteasome substrate suc llvy amc  (Boston Biochem)

Journal: Toxicology

Article Title: An adverse outcome pathway-based approach to assess the neurotoxicity by combined exposure to current-use pesticides.

doi: 10.1016/j.tox.2023.153687

Figure Lengend Snippet: Fig. 4. Evaluation of the effects of pesticides using the proteasome activities as endpoints. Cells were exposed to each pesticide (100 µM) or MG-132 (1 µM) as a positive control for 24 h, and then (A) chymotrypsin-like (Suc-LLVY-AMC) and (B) caspase-like (Z-LLE-AMC) proteasome activities were monitored by AMC- conjugated fluorescence substrates. All data are shown as the mean ± SD. * p < 0.05, * * p < 0.01 vs. the control group (one-way ANOVA followed by Dunnett’s post-hoc test).

Article Snippet: The proteasome assay buffer (100 μL) [50 mM HEPES buffer (pH 7.8) containing 10 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 5 mM DTT and 2 mM ATP] containing 2.5 μM of the fluorogenic proteasome substrate Suc-LLVY-AMC (succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, #S-280) and Z-LLE-AMC (Z-Leu-Leu-Glu-7-amino-4methylcoumarin, #S-230; Boston Biochem, Cambridge, MA, USA) were added to the lysate sample (25 μL) in a 96-well black plate and incubated at 37 ◦C for 30 min.

Techniques: Pesticides, Positive Control, Fluorescence, Control

Journal: Current genetics

Article Title: Characterization of the impact of GMP/GDP synthesis inhibition on replicative lifespan extension in yeast

doi: 10.1007/s00294-020-01068-w

Figure Lengend Snippet: A-B. Lifespan curves corresponding to Step 1 (A) and Step 2 (B) of the LPT test for the proteasome pathway of lifespan extension. N = 300 cells for the MPA condition, pooled from three independent experiments of 100 cells each; N = 400 cells for the DMSO condition, pooled from four independent experiments of 100 cells each; N = 400 cells for the guanine condition, pooled from four independent experiments of 100 cells each. C. Proteasome activity for wild-type (BY4741) cells, or ΔUBR2 cells, in the presence or absence of MPA or guanine. N = 3 biological replicates for each condition. Errors bars are standard error of the mean. D. Lifespan curve for a ΔPRE9 strain in the presence or absence of MPA. N = 200 cells for each condition, pooled from two independent experiments of 100 cells each. In (A, B, D), colored numbers next to the lifespan curves indicate the average replicative lifespan values corresponding to the lifespan curve matching its color. E. A schematic model presenting the relationship of longevity interactions described in this paper. The actions of MPA converge on the actions of the proteasome at the level of GMP pathway inhibition. Inhibition of GMP pathway extends lifespan. Proteasome activation also extends lifespan independently of GMP pathway inhibition.

Article Snippet: The fluorogenic proteasome substrate Suc-LLVY-AMC (Bachem I-1395) was added to a final concentration of 100 μM.

Techniques: Activity Assay, Inhibition, Activation Assay