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2 protocols using horseradish peroxidase conjugated anti rabbit immunoglobulin g

1

Curcumin Modulates Macrophage Osteogenic Differentiation

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Curcumin and lipopolysaccharide (LPS, Escherichia coli O127:B8; purity 99%) were purchased from Sigma-Aldrich (United States). Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (United States). Alkaline Phosphatase (ALP) Assay Kit was purchased from Beyotime (Shanghai, China). Alizarin Red S was purchased from Cyagen (United States). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Boster (Wuhan, China). The primary antibodies for runt-related transcription factor 2 (Runx-2), osteocalcin (OCN), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology, Inc. (United States). The secondary antibodies (horseradish peroxidase-conjugated anti-rabbit immunoglobulin G) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States). The fluorescence secondary antibody for TRITC (Tetramethyl Rhodamin Isothiocyanate) AffiniPure Goat Anti-Rabbit IgG was purchased from EarthOx (San Francisco, CA, United States). The DAPI (4′,6-Diamidino-2-Phenylindole) Staining Kit was obtained from Boster (Wuhan, China). Antibody specific for flow cytometry, including CCR7-PE (phycoerythrin) and CD206-APC (allophycocyanin), were purchased from Biolegend (San Diego, CA, United States).
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2

Western Blot Antibody Reagents

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Antibodies against phospho-p70 S6 kinase (Thr389), p70 S6 kinase, phospho-S6 (Ser235/236), S6, LC3B and α-tubulin were purchased from Cell Signaling Technology (Danvers, MA, USA; cat. nos. 9205, 9202, 4858, 2217, 2775 and 3873). Antibodies against GS were obtained from EMD Millipore (Danvers, MA, USA; cat. no. MAB302). Antibodies against β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA; cat. no. A5441). Horseradish peroxidase-conjugated anti-mouse immunoglobulin G and horseradish peroxidase-conjugated anti-rabbit immunoglobulin G were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA; cat. nos. sc-2748 and sc-2749).
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