Journal: The Journal of Biological Chemistry

Article Title: Low molecular weight heparin promotes the PPAR pathway by protecting the glycocalyx of cells to delay the progression of diabetic nephropathy

doi: 10.1016/j.jbc.2024.107493

Figure Lengend Snippet: Protein expression changes of FABP1, Acox2, Hmgcs2, and Acaa1b, PLTP. A , immunohistochemical staining of FABP1, Acox2, Hmgcs2, and Acaa1b, PLTP and quantification of average fluorescence intensity (n = 3, 20× magnification, three fields of view were selected for each kidney section for quantification). B , information on changes in abundance of five proteins in label-free proteome quantitation. For comparisons involving multiple groups, one-way ANOVA followed by Dunnett's post hoc test was employed. Significance levels are denoted as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. FAB1, fatty acid–binding protein 1.

Article Snippet: Rabbit monoclonal FABP1 antibody (diluted 1:4000; Arkham) was used as the primary antibody and horseradish peroxidase anti-rabbit IgG (Boster) as the secondary antibody.

Techniques: Expressing, Immunohistochemical staining, Staining, Fluorescence, Quantitation Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Low molecular weight heparin promotes the PPAR pathway by protecting the glycocalyx of cells to delay the progression of diabetic nephropathy

doi: 10.1016/j.jbc.2024.107493

Figure Lengend Snippet: Visualization and fluorescence quantification of HK-2 cell surface HS and intracellular FABP1. A , superpositively charged green fluorescent protein (ScGFP) labels highly negatively charged components on the cell surface. The green color diminished after incubation with heparinase ( bottom ), suggesting that HS is the dominant negatively charged species in HK-2. The scale bar represents 20 μm. B , visualization and fluorescence quantification of HS on the cell surface after high-glucose-high-fat treatment and high-glucose-high-fat + LMWH treatment with HK-2. The scale bar represents 20 μm. C , results of quantification of HS on the surface of HK-2 cells by LC-MRM (n = 3). D , composition of HS disaccharides on the surface of HK-2 cells (n = 3, ΔIS: ΔGlcA2S-GlcNS6S, ΔIIS: ΔGlcA-GlcNS6S, ΔIIIS: ΔGlcA2S-GlcNS, ΔIVS: ΔGlcA-GlcNS, ΔIA: ΔGlcA2S-GlcNAc6S, ΔIIA: ΔGlcA-GlcNAc6S, ΔIIA: ΔGlcA2S-GlcNAc6S, ΔIIIA: ΔGlcA2S-GlcNAc, ΔIVA: ΔGlcA-GlcNAc). E , visualization and fluorescence quantification of HK-2 endocytosis FABP1. The change of red fluorescence revealed that LMWH could reverse the endocytosis of FABP1 by HK-2 in a high-glucose-high-fat environment to a certain extent (n = 3, three fields of view were selected and the fluorescence of all cells in each field of view was quantified). The scale bar represents 20 μm. F , assess the effect of altered intracellular FABP1 levels on PPAR signaling using luciferase reporter assays. G , Western blot analysis of target gene proteins downstream of PPAR. H , analysis of the effect of high-glucose-high-fat on PPAR activation. The data from the two cohorts were subjected to analysis using the independent samples t test. For comparisons involving multiple groups, one-way ANOVA followed by Dunnett's post hoc test was employed. Significance levels are denoted as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. FAB1, fatty acid–binding protein 1; HS, heparan sulfate; LC, liquid chromatography; LMWH, low molecular weight heparin; MRM, multiple reaction monitoring; PPAR, peroxisome proliferator–activated receptor.

Article Snippet: Rabbit monoclonal FABP1 antibody (diluted 1:4000; Arkham) was used as the primary antibody and horseradish peroxidase anti-rabbit IgG (Boster) as the secondary antibody.

Techniques: Fluorescence, Incubation, Luciferase, Western Blot, Activation Assay, Binding Assay, Liquid Chromatography, Molecular Weight, Targeted Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Low molecular weight heparin promotes the PPAR pathway by protecting the glycocalyx of cells to delay the progression of diabetic nephropathy

doi: 10.1016/j.jbc.2024.107493

Figure Lengend Snippet: Effect of silencing FABP1 on apoptosis of HK-2 cells in a high-glucose-high-fat environment. For comparisons involving multiple groups, one-way ANOVA followed by Dunnett's post hoc test was employed. Significance levels are denoted as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. FAB1, fatty acid–binding protein 1.

Article Snippet: Rabbit monoclonal FABP1 antibody (diluted 1:4000; Arkham) was used as the primary antibody and horseradish peroxidase anti-rabbit IgG (Boster) as the secondary antibody.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Low molecular weight heparin promotes the PPAR pathway by protecting the glycocalyx of cells to delay the progression of diabetic nephropathy

doi: 10.1016/j.jbc.2024.107493

Figure Lengend Snippet: Validation of FABP1–HS interactions and characterization of oligosaccharide structures involved in the interactions. A , BLI. B , FABP1 affinity chromatography separated affinity LMWH and SEC analysis affinity LMWH polymerization degree changes. C , schematic of complete enzymatic hydrolysis and HONO degradation of LMWH. D , comparison of the relative content of disaccharides after HONO degradation of LMWH-dp8 and affinity-dp8. E , comparison of the relative content of disaccharides after complete enzymatic hydrolysis of LMWH-dp8 and affinity-dp8. F , TOP20 sequence obtained by Sep-GAG software. G , SAX chromatogram of affinity-dp8. H , MS/MS sequencing results of the five major components of affinity-dp8. I , Sep-GAG combined with MS/MS sequencing to obtain sequences of the five components of affinity-dp8. BLI, biolayer interferometry; FAB1, fatty acid–binding protein 1; GAG, glycosaminoglycan; HONO, nitrous acid; HS, heparan sulfate; LMWH, low molecular weight heparin; MS/MS, tandem mass spectrometry; SAX, strong anion exchange chromatography; SEC, size-exclusion chromatography.

Article Snippet: Rabbit monoclonal FABP1 antibody (diluted 1:4000; Arkham) was used as the primary antibody and horseradish peroxidase anti-rabbit IgG (Boster) as the secondary antibody.

Techniques: Biomarker Discovery, Affinity Chromatography, Comparison, Sequencing, Software, Tandem Mass Spectroscopy, Binding Assay, Molecular Weight, Mass Spectrometry, Chromatography, Size-exclusion Chromatography

Journal: The Journal of Biological Chemistry

Article Title: Low molecular weight heparin promotes the PPAR pathway by protecting the glycocalyx of cells to delay the progression of diabetic nephropathy

doi: 10.1016/j.jbc.2024.107493

Figure Lengend Snippet: Molecular docking of dp6 and FABP1. Left panel shows the complex of the FABP1 (shown in part) and the hexasaccharide, visualized using AutoDock simulation. The right panel demonstrates the contributions of the protein and oligosaccharide binding motifs and the types of interactions ( orange dashed arrows represent electrostatic attractions, and green dashed arrows represent hydrogen bonds). FAB1, fatty acid–binding protein 1.

Article Snippet: Rabbit monoclonal FABP1 antibody (diluted 1:4000; Arkham) was used as the primary antibody and horseradish peroxidase anti-rabbit IgG (Boster) as the secondary antibody.

Techniques: Binding Assay