All quantification methods were detailed in our previous study21 (link). Briefly, the QPLEXTM kit utilized Quantamatrix’s multiplex diagnostics platform (QMAP; Quantamatrix Inc., Seoul, Republic of Korea)29 . First, human plasma samples (singular) were diluted in diluent buffer and incubated with the coded beads and biotin-conjugated detection antibodies (angiotensin-converting enzyme, ACE, DY929, R&D Systems, Minneapolis, USA; galectin-3 binding protein, LGALS3BP, DY2226, R&D Systems; periostin, POSTN, DY3548B, R&D Systems; Aβ1−40, 014-26923, Wako, Japan). The immunocomplexes were washed twice with washing buffer at a Biotek-510 magnetic wash station (Biotek, VT, USA). Diluted R-phycoerythrin-conjugated streptavidin was added to each well. After three washes, the immunocomplexes were resuspended in 100 μl of washing buffer by tapping and automatically analyzed using the QMAPTM system. The intra/interassay coefficients of variation and limits of detection were as follows: ACE, intra: 4.9%, inter: 5.1%, 0.22 ng/ml; LGALS3BP, intra: 1.1%, inter: 5.7%, 0.04 ng/ml; POSTN, intra: 9.0%, inter: 6.0%, 0.034 ng/ml; and Aβ1−40, intra: 5.3%, inter: 3.9%, 0.50 pM.
Lgals3bp
Manufactured by R&D Systems
Sourced in United States
LGALS3BP is a protein that functions as a lectin-binding protein. It is involved in cell-cell and cell-matrix interactions.
Automatically generated - may contain errors
Lab products found in correlation
Ca19 9, by Abcam (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Aβ1 40, by Fujifilm (1 mentions)
Dy3548b, by R&D Systems (1 mentions)
P egfr, by Abcam (1 mentions)
Chi3l1, by R&D Systems (1 mentions)
Dc3l10, by R&D Systems (1 mentions)
Dmp900, by R&D Systems (1 mentions)
Mmp 9, by R&D Systems (1 mentions)
P egfr, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
5 protocols using lgals3bp
1
Quantitative Biomarker Profiling using QPLEX
2
Protein Extraction and Western Blot Analysis
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Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Lysates were centrifuged at 14,000 rpm for 15 min, and the supernatant was collected. Subsequently, the proteins were separated via sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. Immunoblotting was performed using antibodies against LGALS3BP (R&D systems), p-EGFR (cell signaling), EGFR (cell signaling), CA19-9 (Abcam), and β-actin (Santa Cruz Biotechnology).
3
Plasma Protein Measurement Protocols
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In the Bruneck year 2000 evaluation and the SAPHIR study, the following proteins were measured by ELISA in plasma samples according to the manufacturers’ instructions: MMP9 from R&D Systems (DMP900); CHI3L1 from R&D Systems (DC3L10); LGALS3BP from R&D Systems (DGBP30); S100A8/A9 from BMA Biomedicals. Additional plasma proteins were analyzed using proximity extension assays (CVD I and Inflammation I panels, Olink) as previously published (15 (link)).
4
Protein Expression Analysis of Key Markers
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Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Lysates were centrifuged at 14,000 rpm for 15 min, and the supernatant was collected. Subsequently, the proteins were separated via SDS-polyacrylamide gel electrophoresis. Immunoblotting was performed using antibodies against LGALS3BP (R&D systems), P-EGFR (Cell Signaling), EGFR (Cell Signaling), CA19-9 (Abcam), and β-actin (Santa Cruz Biotechnology).
5
Western Blotting Analysis of 17-AAG-Induced LGALS3BP Secretion
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Western blotting analysis was performed as described previously (37) . Briefly, the cells were lysed in modified RIPA buffer (1 mol/L Tris-HCl, pH 7.4, 5 mol/L NaCl, 0.5 mol/L EDTA, 1 mol/L NaF, 2 mol/L Na3VO4 protease inhibitor, 10% deoxycholate, 0.1% NP-40, and 0.1% Triton X-100) as described elsewhere (38) . Equal amounts of protein (20-50 mg) were loaded in 8% or 10% SDS-PAGE gels and transferred by blotting to polyvinylidene fluoride membranes. Western blotting antibodies were as follows: LGALS3BP (R&D Systems), Akt, pAkt, ERK, pERK, PARP, caspase-3 (Cell Signaling Technology), and GAPDH (EMD Millipore). Detection was performed using chemiluminescence (Amersham) according to the manufacturer's instructions. Blots were visualized by autoradiography. 17-AAG-mediated secretion of LGALS3BP into culture supernatant was evaluated using Western blotting analysis. The cells were cultured with serial dilution of 17-AAG or medium only. The cell culture supernatants were collected at the indicated times and centrifuged at 1.3 Â 10 4 rpm for 20 minutes at 4 C. Western blotting analysis was performed on the collected supernatants.
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