mouse anti vdbp  (Santa Cruz Biotechnology)

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 3. Legumain is required for VDBP processing and regulation. (A) Purified VDBP from human plasma (1.9 µM) was incubated in legumain assay buffer (pH 5.8) at 37 ◦C with or without purified active bovine legumain (2 µM) for 5 h before gel electrophoresis and immunoblotting of VDBP (n = 1). (B–H) Wild-type (Lgmn+/+) and legumain-deficient (Lgmn−/−) mice were treated with 50 µg/kg 25(OH)D3 (n = 6–7) or an equal volume vehicle (n = 7, control) subcutaneously every two to three days (four times in total). Tissues were harvested 24 h after the final injection (day 8). (B) One representative immunoblot of VDBP and GAPDH (housekeeping) in kidney and liver (n = 4). (C–F) Quantification of VDBP immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH in immunoblots represented in (B) (n = 4). (C) Hepatic VDBP 45 kDa immunoband. (D) Renal VDBP 45 kDa immunoband. (E) Hepatic VDBP 55 kDa immunoband. (F) Renal VDBP 55 kDa immunoband. (G) Plasma VDBP concentration (µg/mL) was measured by ELISA (n = 6–7). (H) Hepatic VDBP mRNA expression relative to the geometric mean of CT values of four house- keeping controls (2−∆∆CT, n = 5). (C–H) Data represent mean ± SEM. Two-way ANOVA. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. different genotype, same treatment. Numbers (n) represent individual biological replicates.

Article Snippet: The membranes were blocked for 1 h at room temperature with Odyssey® Blocking Buffer and probed with polyclonal goat anti-human legumain (1:200, R&D Systems, Minneapolis, MN, USA, Catalog # AF2199, RRID: AB_416565), polyclonal rabbit anti-human/mouse VDBP (1:500, Bio-Techne, Minneapolis, MN, USA, Catalog # NBP1-88027, RRID: AB_11023579), monoclonal mouse anti-human VDBP (1:500, R&D Systems, Catalog # MAB3778, RRID: AB_2232276), monoclonal mouse anti-human GAPDH antibody (1:10,000, Santa Cruz Biotechnology Inc., Dallas, TX, USA, Catalog # sc-47724, RRID: AB_627678), or monoclonal mouse anti-human GAPDH (1:10,000, R&D Systems, Catalog # MAB5718, RRID: AB_10892505) antibody in Tris-buffered saline containing 0.1% Tween 20 (T-TBS) overnight at 4 ◦C.

Techniques: Purification, Clinical Proteomics, Incubation, Nucleic Acid Electrophoresis, Western Blot, Control, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 5. Graphical representation of the suggested interplay between vitamin D and legumain. Left panel: Vitamin D (VD3) promotes legumain expression and activity through transcriptional upregulation of the legumain gene (LGMN). The free fraction of circulating VD3 metabolites diffuse through plasma membranes. 25-hydroxyvitamin D (25(OH)D3) is hydroxylated by 1α-hydroxylase (CYP27B1), forming the active metabolite 1α,25-dihydroxyvitamin D (1,25(OH)2D3). 1,25(OH)2D3 binds to the nuclear vitamin D receptor (VDR) and promotes transcription of legumain (LGMN). Synthesized prolegumain is either sorted and activated in the endolysosomal system or released to the extracellular environment. Right panel: In the proximal tubular epithelium, 25(OH)D3 bound to vitamin D binding protein (VDBP) is internalized from the tubular lumen through a megalin/cubilin- mediated process. The vitamin D metabolite is released, enabling subsequent hydroxylation by 1α-hydroxylase (CYP27B1) or 24-hydroxylase (CYP24A1), and VDBP is cleaved by legumain in the endolysosomal system. VDBP cleavage by legumain might be important in controlling the systemic level of vitamin D metabolites. Created with BioRender.com (accessed on 11 December 2023).

Article Snippet: The membranes were blocked for 1 h at room temperature with Odyssey® Blocking Buffer and probed with polyclonal goat anti-human legumain (1:200, R&D Systems, Minneapolis, MN, USA, Catalog # AF2199, RRID: AB_416565), polyclonal rabbit anti-human/mouse VDBP (1:500, Bio-Techne, Minneapolis, MN, USA, Catalog # NBP1-88027, RRID: AB_11023579), monoclonal mouse anti-human VDBP (1:500, R&D Systems, Catalog # MAB3778, RRID: AB_2232276), monoclonal mouse anti-human GAPDH antibody (1:10,000, Santa Cruz Biotechnology Inc., Dallas, TX, USA, Catalog # sc-47724, RRID: AB_627678), or monoclonal mouse anti-human GAPDH (1:10,000, R&D Systems, Catalog # MAB5718, RRID: AB_10892505) antibody in Tris-buffered saline containing 0.1% Tween 20 (T-TBS) overnight at 4 ◦C.

Techniques: Expressing, Activity Assay, Clinical Proteomics, Synthesized, Binding Assay