Journal: bioRxiv

Article Title: Boosting Proteasome Activity: A Novel Mechanism of NMDAR Blockers Against Neurodegeneration

doi: 10.1101/2024.08.20.608787

Figure Lengend Snippet: (A) Effects of RPN13 inhibition by RA190 on p21 levels and the corresponding influence of ketamine in RA190 (1µM, 12 h incubation) pre-treated conditions. (B and C) Role of RA190 in chymotrypsin-like activity and ketamine’s intervention in these conditions. (D) RA190’s elevation of ubiquitinated proteins contrasted with ketamine’s reduction effect. (E) IU1’s effect on p21 protein levels and the modifying role of ketamine alongside USP14 inhibition. (F) Changes in USP14 protein levels with overexpression or silencing techniques. (G) How USP14 expression levels affect p21 and p53 proteins and ketamine’s influence under these conditions. (H, I) Impact of USP14 expression alterations on chymotrypsin-like activity and modifications by ketamine. (J) Dose-dependent effects of the PSMD14 inhibitor gliotoxin on p21 levels were examined, alongside the effects of ketamine on p21 with and without gliotoxin pre-treatment, with evidence that gliotoxin inhibits 20S proteasome chymotrypsin activity at high doses. p21 levels were analyzed via western blot at two time points of signal exposure. (K and L) Ketamine’s effects on chymotrypsin activity were evaluated with and without 5 µM gliotoxin. ( M) At 25 µM gliotoxin, ketamine fails to decrease p21 levels while reducing p53 levels. Due to this discrepancy, the Western blot was repeated multiple times and the result was ultimately confirmed by using p21 and p53 antibodies concurrently on the same membrane (N and O) Chymotrypsin activity in response to ketamine was assessed with and without 25 µM gliotoxin. Results are presented as the mean ± SEM, based on 3 to 5 technical replicates from each of n = 3 independent experiment. *Asterisks denote significance levels: *p < 0.05, **p < 0.01, ***p < 0.001. P values were calculated using a two-tailed unpaired t-test to compare the control group with the individual chemical effect or USP14 knockdown cells.

Article Snippet: We measured proteasome activity with the Enzo Life Sciences’ 20S assay kit, applying isolated and purified proteasomes from human erythrocytes.

Techniques: Inhibition, Incubation, Activity Assay, Over Expression, Expressing, Western Blot, Membrane, Two Tailed Test, Control, Knockdown

Journal: EMBO Molecular Medicine

Article Title: Activin receptor-like kinase5 inhibition suppresses mouse melanoma by ubiquitin degradation of Smad4, thereby derepressing eomesodermin in cytotoxic T lymphocytes

doi: 10.1002/emmm.201302524

Figure Lengend Snippet: Source data is available for this figure in the Supporting Information. PLA (red) show the close proximity between ubiquitin and Smad4 in the dLN cells co-stained with anti-CD8 (green) (scale bars: 5 µm, 50 µm). Graphs show mean PLA signals in nuclei (black) and cytoplasms (white) quantified using BlobFinder software. Upper panel shows endogenous ubiquitinated Smad4 and lower panel shows ubiquitinated proteins in CD8 + and CD8 − dLN cells. Ubiquitinated proteins were captured using an UbiQapture™-Q kit and blotted with anti-Smad4 or anti-ubiquitin antibody. Molecular weight of Smad4 is 70 kD. Western blots show Smads in CD4 + and CD8 + cells stimulated with anti-CD3/CD28 with/without EW-7197 and/or MG-132 for 3 days. IP-Western blot shows endogenous ubiquitinated Smad4 in CD4 + and CD8 + cells stimulated with anti-CD3/CD28 with/without EW-7197 and/or MG-132 for 3 days. Representative immunohistochemistry sections of inoculated melanomas (scale bar: 100 µm). Graph shows the subcellular distributions of Smad4 expression in melanoma cells calculated by ImageJ software. The expression ratios of nucleus to cytoplasm are shown. Smad4 protein in B16 cells was detected by PLA (red; scale bars: 10 µm; left). The expression ratios of nucleus to cytoplasm are shown. Graph shows the subcellular distributions of Smad4 in B16 cells. Western blots show Smads in B16 cells cultured with EW-7197 with or without TGF-β1 (right).

Article Snippet: Ubiquitinated Smad4 in CD8 + dLN cells was detected using an UbiQapture-Q kit (Enzo Life Sciences) according to the manufacturer's protocol.

Techniques: Staining, Software, Molecular Weight, Western Blot, Immunohistochemistry, Expressing, Cell Culture