Journal: Shock (Augusta, Ga.)

Article Title: RESVERATROL DOWNREGULATES BIOMARKERS OF SEPSIS VIA INHIBITION OF PROTEASOME’S PROTEASES

doi: 10.1097/SHK.0000000000001080

Figure Lengend Snippet: Measurement of Proteasome Activities. Dose-dependent inhibition of purified human proteasome or immunoproteasome protease activities using resveratrol (0.1 μM – 100 μM), and ONX-0914 (0.01 μM – 80 μM). Proteasome activities were measured at excitation 345 and emission 445 wavelengths after addition of peptide fluorogenic substrates using Cytation 3 Plate Multi-mode reader. Data are shown as means ± SE (n=3) and each reaction was performed in triplicates. Results were plotted as percentage inhibition compared with vehicle (DMSO) control.

Article Snippet: Purified human erythrocyte 20S proteasome and peripheral blood monocyte 20S immunoproteasomes (R&D Systems) were assayed for different peptidase activities using fluorogenic tri-and tetra-peptide substrates coupled to AMC.

Techniques: Inhibition, Purification, Control

Journal: Shock (Augusta, Ga.)

Article Title: RESVERATROL DOWNREGULATES BIOMARKERS OF SEPSIS VIA INHIBITION OF PROTEASOME’S PROTEASES

doi: 10.1097/SHK.0000000000001080

Figure Lengend Snippet: Measurement of Cellular Proteasome Activities. Dose dependent effect of resveratrol on cellular proteasome activity. THP1 cells at 1×104 cells/well were treated with different concentrations of resveratrol (0.1 μM – 100 μM) and incubated at 37°C and 5% CO2, for 30 min. The relative luminescence units (RLU) of assays were read in a Cytation 3 Plate Multi-mode reader. Data are shown as means ±SE (n=3) and each reaction was performed in triplicates. Results were plotted as percentage inhibition compared with vehicle (DMSO) control.

Article Snippet: Purified human erythrocyte 20S proteasome and peripheral blood monocyte 20S immunoproteasomes (R&D Systems) were assayed for different peptidase activities using fluorogenic tri-and tetra-peptide substrates coupled to AMC.

Techniques: Activity Assay, Incubation, Inhibition, Control

Journal: Shock (Augusta, Ga.)

Article Title: RESVERATROL DOWNREGULATES BIOMARKERS OF SEPSIS VIA INHIBITION OF PROTEASOME’S PROTEASES

doi: 10.1097/SHK.0000000000001080

Figure Lengend Snippet: Effects of resveratrol (80 μM) or ONX-0914 (0.25 μM) on proteasome subunits gene expression using CD14+ human blood monocytes. CD14+ monocytes (2×106 cells/well) were treated for Vehicle, LPS, Res, Res+LPS, ONX and ONX+LPS conditions, as described in the methods section. Gene expression of proteasome subunits PSMB5 (X), PSMB6 (Y), PSMB7 (Z), PSMB8 (LMP7), PSMB9 (LMP2) and PSMB10 (LMP10) were analyzed by qRT-PCR. Data are shown as means ±SE (n=3). *Statistically significant difference of LPS treatment vs veh, Res, Res+LPS, ONX and ONX+LPS (p≤ 0.05).

Article Snippet: Purified human erythrocyte 20S proteasome and peripheral blood monocyte 20S immunoproteasomes (R&D Systems) were assayed for different peptidase activities using fluorogenic tri-and tetra-peptide substrates coupled to AMC.

Techniques: Gene Expression, Quantitative RT-PCR

Journal: medRxiv

Article Title: Elevated plasma Complement Factor H Regulating Protein 5 is associated with venous thromboembolism and COVID-19 severity

doi: 10.1101/2022.04.20.22274046

Figure Lengend Snippet: Platelet activation was measured by surface expression of ( A ) P-selectin, ( B ) activated GP IIb/IIIa or ( C ) CD63, following treatment of platelet rich plasma with different concentrations of ( i ) adenosine diphosphate (ADP) ( ii ) convulxin or ( iii ) TRAP6, following pre-incubation (10 minutes) with recombinant CFHR5, or PBS control. Each experiment is represented by an individual point and paired experiments connected by a dotted line. *p<0.05 **p<0.01 ***p<0.001 (p for trend bottom right).

Article Snippet: Platelet-rich plasma (PRP) or isolated platelets were incubated with recombinant CFHR5 in PBS (6 μg/ml, 3845-F5, R&D systems) or PBS alone for 10 minutes before treatment with varying concentrations of ADP (1-5 μM), TRAP-6 (3-15 μM) or convluxin (1-6 ng/ml) for 15 minutes.

Techniques: Activation Assay, Expressing, Clinical Proteomics, Incubation, Recombinant, Control

Journal: bioRxiv

Article Title: Protein SUMOylation promotes cAMP-independent EPAC1 activation

doi: 10.1101/2024.01.08.574738

Figure Lengend Snippet: Levels of cellular EPAC1 PTM probed by immunoblotting using anti-EPAC1 antibody in HUVEC (A) and HEK293/EPAC1-Flag (B) in response to heat shock as a function of time. (C) Levels of cellular EPAC1 PTM in HEK293/EPAC1-EYFP cells probed by immunoblotting using anti-EPAC1 antibody, with and without heat shock (30 min) and with or without SENP1 (220 nM) treatment at 37 °C for 20 min. Similar results were obtained from at least three independent experiments.

Article Snippet: To test if EPAC1 PTM was sensitive to SUMO-specific deconjugating enzyme, heat shock treated cells were lysed with 1× cell lysis buffer without 20 mM NEM and then incubated with 220 nM recombinant Human Sentrin-specific protease 1 (SENP1) catalytic domain (R&D Systems, Catalog no. E-700) at 37 °C for 20 min before immunoblotting analysis.

Techniques: Western Blot

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Molecular characterization of a precision-cut rat liver slice model for the evaluation of antifibrotic compounds

doi: 10.1152/ajpgi.00281.2018

Figure Lengend Snippet: ELISA analysis of secreted biomarkers. SB525334, nintedanib, and sorafenib suppressed secretion of hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) in the conditioned medium after 48 h of incubation. Data are means ± SE; n = 3 rats for both sham and bile duct ligation (BDL); n = 8–10 liver slices for each condition. **P < 0.01 and ***P < 0.001.

Article Snippet: Secreted biomarkers including procollagen I, HA, IGFBP5, and WISP1 in the conditioned medium were measured using procollagen I ( {"type":"entrez-nucleotide","attrs":{"text":"Ab210579","term_id":"70570352","term_text":"AB210579"}} Ab210579 ; Abcam, Cambridge, UK), hyaluronan quantikine (DHYAL0; R&D Systems, Minneapolis, MN), IGFBP5 ( {"type":"entrez-nucleotide","attrs":{"text":"Ab208345","term_id":"76667415","term_text":"AB208345"}} Ab208345 ; Abcam), and WISP1 quantikine (MWSP10; R&D Systems) ELISA kits following manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Ligation

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Molecular characterization of a precision-cut rat liver slice model for the evaluation of antifibrotic compounds

doi: 10.1152/ajpgi.00281.2018

Figure Lengend Snippet: The small molecule αV integrins inhibitor CWHM12 attenuated fibrotic phenotype in fibrotic precision-cut liver tissue slices (PCLSs) after 48 h of incubation. A: CWHM12 suppressed expression of a panel of fibrotic genes. B: CWHM12 decreased secretion of procollagen I, hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) in the conditioned medium. Data are means ± SE; n = 3 rats for both sham and bile duct ligation (BDL); n = 6–8 liver slices for each condition. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Secreted biomarkers including procollagen I, HA, IGFBP5, and WISP1 in the conditioned medium were measured using procollagen I ( {"type":"entrez-nucleotide","attrs":{"text":"Ab210579","term_id":"70570352","term_text":"AB210579"}} Ab210579 ; Abcam, Cambridge, UK), hyaluronan quantikine (DHYAL0; R&D Systems, Minneapolis, MN), IGFBP5 ( {"type":"entrez-nucleotide","attrs":{"text":"Ab208345","term_id":"76667415","term_text":"AB208345"}} Ab208345 ; Abcam), and WISP1 quantikine (MWSP10; R&D Systems) ELISA kits following manufacturer's instructions.

Techniques: Incubation, Expressing, Binding Assay, Ligation