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2 protocols using alkaline phosphatase alp assay kit

1

Osteoclast Differentiation Regulatory Factors

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Recombinant human BMP2, M-CSF, and RANKL were purchased from PeproTech (Rocky Hill, NJ, USA). Phospho-specific (Ser151) and pan-CRTC1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). HG-9-91-01 was purchased from MedChemExpress (Princeton, NJ, USA). Antibodies against SIK1, Id1, Lamin B, and α-tubulin, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). OCN antibody was acquired from Abcam (Cambridge, UK). Anti-β-actin and anti-HA were acquired from Sigma-Aldrich (St. Louis, MO, USA). HRP Anti-Rabbit IgG (Peroxidase) Polymer Detection Kit was purchase from Vector Laboratories (Burlingame, CA, USA). Additionally, an alkaline phosphatase (ALP) assay kit was purchased from Takara Bio Inc. (Ohtsu, Japan), while an ALP staining kit, tartrate-resistant acid phosphatase (TRAP) staining kit, β-glycerophosphate, and ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse RANKL and OPG ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). pcDNA3-SIK1-HA, pcDNA3-SIK1-FLAG, and pcDNA3-SIK1-T182A-FLAG plasmids were as previously described39 (link). pGL-Id1 (−1231/+88) plasmid was provided by Dr. Korchynskyi (National Academy of Sciences in Ukraine)40 (link). siRNA oligonucleotides for control, SIK1, SIK2, SIK3, or CRTC1 were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
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2

Regulation of Osteoblast Differentiation

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Recombinant mouse S100A4 was purchased from Prospec (East Brunswick, NJ, USA). Phosphospecific antibodies against IκB kinase (IKK)α/β (Ser176/180), IκB (Ser32), p65 (Ser536), IKKα/β, IκB, and p65 were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against osterix, laminB, and p65 were acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-β-actin (AC-74) was purchased from Sigma-Aldrich (St. Louis, MO, USA), anti-Runx2 from MBL International (Woburn, MA, USA), the alkaline phosphatase (ALP) assay kit from Takara Bio Inc. (Fukui, Japan). β-Glycerophosphate, cetylpyridium chloride, and ascorbic acid from Sigma-Aldrich, Bay11-7082 from Alexis Biochemicals (Grunberg, Germany), and the Cell Counting Kit-8 (CCK-8) from Dojindo (Kumamoto, Japan).
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