Journal: Antioxidants

Article Title: Olive Leaf Extract Added to Losartan Treatment Improved Klotho/Wnt/β-Catenin Signaling in Hypertensive Rats with Focal Segmental Glomerulosclerosis

doi: 10.3390/antiox15010146

Figure Lengend Snippet: Effects of L, L+T and L+O on 6–nitrotryptophan, Klotho, Wnt4, β–catenin, PAI–1 and fibronectin protein expressions in the kidney of SHR with ADR-induced FSGS. The analysis of 6–nitrotryptophan/actin ( A ), Klotho/actin ( B ), Wnt4/GAPDH ( C ), β–catenin/GAPDH ( D ), PAI–1/GAPDH ( E ), fibronectin/GAPDH ( F ) protein expressions and representative Western blots ( G ). Values represent mean ± SEM of three to four independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. SHC; # p < 0.05 and ### p < 0.001 vs. SHADR; $$ p < 0.01 and $$$ p < 0.001 vs. SHADR+L; and &&& p < 0.001 vs. SHADR+L+T. SHC, control group; SHADR, model group; L, losartan; T, tempol; and O, olive leaf extract.

Article Snippet: The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBS-Tween) and then incubated with the following primary antibodies: 6-nitrotryptophan (1:1000, ab243072, Abcam, Cambridge, UK), Klotho (1:1000, NBP1-76511, Novus biologicals, LCC, USA), Wnt-4 (1:2000, NBP2-20909, Novus biologicals, LCC, Centennial, CO, USA), β-catenin (1:300, AF1329, R&D Systems, Inc., Minneapolis, MN, USA), PAI-1 (1:1000, NBP1-19773, Novus biologicals, LCC, Centennial, CO, USA), fibronectin (1:1000, ab2413, Abcam, Cambridge, UK), actin (1:500, A5060, Sigma-Aldrich, MO, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2500, ab9485, Abcam, Cambridge, UK).

Techniques: Western Blot, Control

Journal: iScience

Article Title: Human monocyte-derived macrophages shift subcellular metalloprotease activity depending on their activation state

doi: 10.1016/j.isci.2024.111171

Figure Lengend Snippet: Proteases and protease inhibitors/activators are increased in the cell culture supernatants and cell lysate of M(IL-4) compared to M(LPS/IFN-γ) (A) Protease and protease regulators detected in cell culture supernatants of M(M-CSF), M(LPS/IFN-γ), and M(IL-4) by proteome profiler array analysis. Expression was measured in comparison to the internal array controls and depicted as a scale bar from 0% to 100%. White squares indicate undetectable expression levels. (B) Quantification of secreted proteases and regulators in cell culture supernatants of M(M-CSF), M(LPS/IFN-γ), and M(IL-4) by gelatin in gel zymography (for MMP-9 protein levels) and ELISA (for MMP-12, cathepsin D, TIMP-1, TIMP-2, serpin E1, cystatin C). Each data point represents cells from a different donor (at least n = 7). Error bars are mean ± SEM. p values less than 0.05 were considered significant (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns non-significant). (C) Quantification of secreted MMP-9/TIMP-1 complex levels in cell culture supernatants of M(M-CSF), M(LPS/IFN-γ), and M(IL-4) by ELISA. Each data point represents cells from a different donor ( n = 9). Error bars are mean ± SEM. p values less than 0.05 were considered significant (∗ p < 0.05; ns non-significant). (D) Analysis of proteases and protease regulators in cell lysates of M(M-CSF), M(LPS/IFN-γ), and M(IL-4), measured using proteome profiler arrays. Expression was measured in comparison to the internal array controls and depicted as a scale bar from 0% to 100%. White squares indicate undetectable expression levels. (E) Quantification of cell-associated proteases and regulators of M(M-CSF), M(LPS/IFN-γ), and M(IL-4) by gelatin in gel zymography (for MMP-9 protein levels), ELISA (for MMP-12, ADAM-9, TIMP-2, serpin E1, and cystatin C) and flow cytometry (for EMMPRIN). Each data point represents cells from a different donor (at least n = 8). Error bars are mean ± SEM. p values less than 0.05 were considered significant (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns non-significant). (F) MMP-9 protein quantification by gelatin in gel zymography in the cell lysate of M(M-CSF), M(LPS/IFN-γ), and M(IL-4). See also Figures S2–S5 .

Article Snippet: MMP-12 (Human MMP-12 ELISA, Cat # EH327RB, Invitrogen), ADAM-9 (Human ADAM-9 DuoSet ELISA, Cat # DY939, R&D Systems), TIMP-1 (Human TIMP-1 DuoSet ELISA, Cat # DY970, R&D Systems), TIMP-2 (Human TIMP-2 DuoSet ELISA, Cat # DY971, R&D Systems), MMP-9/TIMP-1 complex (Human MMP-9/TIMP-1 DuoSet ELISA, Cat # DY1449, R&D Systems), Cystatin C (Human Cystatin C DuoSet ELISA, Cat # DY1196, R&D Systems), Cathepsin D (Human Cathepsin D DuoSet ELISA, Cat # DY1014, R&D Systems), Serpin E1 (Human Serpin E1 DuoSet ELISA, Cat # DY1786, R&D Systems), Tumor Necrosis Factor alpha (TNF-α) (Human TNF-alpha DuoSet ELISA, Cat # DY210, R&D Systems), IL-1β (Human IL-1 beta DuoSet ELISA, Cat # DY201, R&D Systems), IL-6 (Human IL-6 DuoSet ELISA, Cat # DY206, R&D Systems) and CCL-18 (Human CCL18/PARC DuoSet ELISA, Cat # DY394, R&D Systems) concentrations were measured by sandwich ELISA according to the manufacturer’s protocols.

Techniques: Cell Culture, Expressing, Comparison, Zymography, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: iScience

Article Title: Human monocyte-derived macrophages shift subcellular metalloprotease activity depending on their activation state

doi: 10.1016/j.isci.2024.111171

Figure Lengend Snippet:

Article Snippet: MMP-12 (Human MMP-12 ELISA, Cat # EH327RB, Invitrogen), ADAM-9 (Human ADAM-9 DuoSet ELISA, Cat # DY939, R&D Systems), TIMP-1 (Human TIMP-1 DuoSet ELISA, Cat # DY970, R&D Systems), TIMP-2 (Human TIMP-2 DuoSet ELISA, Cat # DY971, R&D Systems), MMP-9/TIMP-1 complex (Human MMP-9/TIMP-1 DuoSet ELISA, Cat # DY1449, R&D Systems), Cystatin C (Human Cystatin C DuoSet ELISA, Cat # DY1196, R&D Systems), Cathepsin D (Human Cathepsin D DuoSet ELISA, Cat # DY1014, R&D Systems), Serpin E1 (Human Serpin E1 DuoSet ELISA, Cat # DY1786, R&D Systems), Tumor Necrosis Factor alpha (TNF-α) (Human TNF-alpha DuoSet ELISA, Cat # DY210, R&D Systems), IL-1β (Human IL-1 beta DuoSet ELISA, Cat # DY201, R&D Systems), IL-6 (Human IL-6 DuoSet ELISA, Cat # DY206, R&D Systems) and CCL-18 (Human CCL18/PARC DuoSet ELISA, Cat # DY394, R&D Systems) concentrations were measured by sandwich ELISA according to the manufacturer’s protocols.

Techniques: Plasmid Preparation, Recombinant, Knock-Out, Blocking Assay, Staining, Antibody Labeling, Reverse Transcription, Lysis, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Fractionation, Cell Culture, Software, Flow Cytometry, Fluorescence, Spectrophotometry