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

Journal: Pharmaceutics

Article Title: Liposomal Formulations Enhance the Anti-Inflammatory Effect of Eicosapentaenoic Acid in HL60 Cells

doi: 10.3390/pharmaceutics14030520

Figure Lengend Snippet: The inhibition of production of NO, ROS, and cytokines by EPA-liposomes. The differentiated HL60 cells were treated with free EPA or EPA-liposomes at the indicated concentrations in the presence of LPS (100 ng/mL) for 24 h, and the NO contents ( A ) in the cell culture media were determined by the Griess agent. ( B ) The ROS levels in the cells were determined by the ROS detection kit. ( C ) The cytokine levels in the cell culture medium were determined by the ELISA kit following the guidance of the manual. The data are expressed as means ± SD. * p < 0.05, ** p < 0.01, n = 3.

Article Snippet: The collected cells were resuspended in a white 96-well plate for ROS measurement using a total ROS detection kit (Enzo Life Sciences, Farmingdale, NY, USA) per the guidance of the manual.

Techniques: Inhibition, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomic Analysis Reveals Important Roles of the Acetylation of ER-Resident Molecular Chaperones for Conidiation in Fusarium oxysporum

doi: 10.1016/j.mcpro.2022.100231

Figure Lengend Snippet: Effects of acetylation on protein function of PDI. A , schematic representation of PDI structure. The architecture of PDI consists of five domains (abb'a'c). The active sites of the a and a' domains are marked with red color . The acetylation site of K70 is marked with blue color. The three-dimensional predicted PDI structure modelled using SWISS-MODEL web server and PYMOL software. B , determination of PDI activity. The recombinant PDI-His protein or its mutant isoforms were collected through Ni-NTA affinity purification. The PDI-GFP protein or its mutant isoforms were immunoprecipitated with anti-GFP antibody agarose beads. 14 μg of different isoforms of PDI proteins were subjected to PDI reductase activity detection according to the PROTEOSTAT PDI assay kit instructions. Results are expressed as relative fluorescence units (RFU). C , immunoblot analysis of PDI stability. The SM of different strains were treated with 10 μΜ CHX for the indicated time. Total proteins were extracted and then subjected to Western blot analysis with anti-GFP or anti-H3 antibody. Numbers below the blots represent the relative protein abundance. anti-H3 immunoblotting was used to show equal loading. D , protein aggregation of SM measured using the PROTEOSTAT Protein Aggregation Assay Kit following the manufacturer's instructions (n = 3). E , effects of PDI inhibitors on F. oxysporum mycelia growth and conidiation. The presence of different letters above the mean values of three replicates indicates a significant difference between different strains and samples ( p < 0.05, ANOVA). CHX, cycloheximide; NM, nonsporulating mycelia; SM, sporulating mycelia.

Article Snippet: For the PDI activity assay, different isoforms of recombinant or immunoprecipitated PDI proteins were quantified to 14 μg using NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific).Then, these proteins were used to measure PDI activity with PROTEOSTAT PDI assay kit (Enzo life sciences) according to the manufacturer’s protocol.

Techniques: Software, Activity Assay, Recombinant, Mutagenesis, Affinity Purification, Immunoprecipitation, Fluorescence, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Quantitative Proteomic Analysis Reveals Important Roles of the Acetylation of ER-Resident Molecular Chaperones for Conidiation in Fusarium oxysporum

doi: 10.1016/j.mcpro.2022.100231

Figure Lengend Snippet: Effects of acetylation on protein function of HSP70 and HSP40. A , schematic of HSP70 structure which consists of an N-terminal nucleotide-binding domain (NBD), a substrate binding domain (SBD), and a C-terminal α-helix domain acting as a lid. The acetylation site of K604 is marked with blue color . B , protein aggregation of SM of the indicated strains. C , schematic of HSP40 structure. The N-terminal J-domain is marked in orange colored box and K32ac site is marked in blue . The three-dimensional predicted full-length HSP70/HSP40 structure was modeled using SWISS-MODEL web server and PYMOL software. D , protein aggregation of SM of the indicated strains measured using the PROTEOSTAT Protein Aggregation Assay Kit following the manufacturer's instructions (n = 3). E , a model for acetylation of the ER-resident molecular chaperones modulating conidiation of F. oxysporum . The presence of different letters above the mean values of three replicates indicates a significant difference between different strains and samples ( p < 0.05, ANOVA).

Article Snippet: For the PDI activity assay, different isoforms of recombinant or immunoprecipitated PDI proteins were quantified to 14 μg using NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific).Then, these proteins were used to measure PDI activity with PROTEOSTAT PDI assay kit (Enzo life sciences) according to the manufacturer’s protocol.

Techniques: Binding Assay, Software