Curcumin and Glucuronide Assays for Inflammatory Pathways

Curcumin (218580100, Fisher; 80.6% curcumin, 13.5% demethoxycurcumin, and 2.4% bisdemethoxycurcumin by weight) and curcuminglucuronide were purchased, with content/purity verified using standard LC/MS methods (see below)^{11 (link),60 (link)} with stock solutions prepared in DMSO. Murine RAW 264.7 cells were obtained from American Type Culture Collection (#TIB-71), ATCC) and used within 10 passages. Primary antibodies against GUSB (ARP44234_T100, Aviva Systems Biology), HPSE (bs-1541R, Bioss USA), KL (ab181373, Abcam) and HRP secondary antibodies (7074, Cell Signaling) were purchased. Phenolphthalein-glucuronide (PhePG; P0501), 4-methylumbelliferyl-glucuronide (4-MUG; M9130), D-Saccharic acid 1,4-lactone monohydrate (saccharolactone, S0375), and RIPA lysis buffer (R0278) were purchased from Sigma Aldrich. Supersignal West Femto ECL (34095) and RestorePlus stripping buffer (46430) were purchased from ThermoFisher. Recombinant human (rh)-GUSB (6144-GH), rh-HPSE (7570-GH), and rh-KL (5334-KL), and recombinant mouse receptor activator of NFkB ligand (RANKL, 462-TEC) were purchased from R&D Systems. Mini-PROTEAN TGX-PAGE gels (4568046) were purchased from BioRad and PVDF membranes (IPFL0010) from Millipore.

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Kunihiro A.G., Luis P.B., Brickey J.A., Frye J.B., Chow H.H., Schneider C, & Funk J.L. (2019). Beta-glucuronidase catalyzes deconjugation and activation of curcumin-glucuronide in bone. Journal of natural products, 82(3), 500-509.

Publication 2019

ARP44234_T100, RIPA lysis buffer Supersignal West Femto ECL RestorePlus stripping buffer RANKL, 462-TEC) Mini-PROTEAN TGX-PAGE gels

4 mug Antibodies Bisdemethoxycurcumin Buffer Curcumin D saccharic acid Demethoxycurcumin Dmso Gels Hpse Human Lactone Ligand Membranes Mouse Nfkb Phenolphthalein glucuronide Pvdf Rankl Raw 264 7 cells Ripa Saccharolactone

Corresponding Organization : University of Arizona

Other organizations : Vanderbilt University

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Variable analysis

independent variables

Curcumin (218580100, Fisher; 80.6% curcumin, 13.5% demethoxycurcumin, and 2.4% bisdemethoxycurcumin by weight)
Curcuminglucuronide

dependent variables

Not explicitly mentioned

control variables

Murine RAW 264.7 cells were obtained from American Type Culture Collection (#TIB-71), ATCC) and used within 10 passages
Primary antibodies against GUSB (ARP44234_T100, Aviva Systems Biology), HPSE (bs-1541R, Bioss USA), KL (ab181373, Abcam) and HRP secondary antibodies (7074, Cell Signaling) were purchased
Phenolphthalein-glucuronide (PhePG; P0501), 4-methylumbelliferyl-glucuronide (4-MUG; M9130), D-Saccharic acid 1,4-lactone monohydrate (saccharolactone, S0375), and RIPA lysis buffer (R0278) were purchased from Sigma Aldrich
Supersignal West Femto ECL (34095) and RestorePlus stripping buffer (46430) were purchased from ThermoFisher
Recombinant human (rh)-GUSB (6144-GH), rh-HPSE (7570-GH), and rh-KL (5334-KL), and recombinant mouse receptor activator of NFkB ligand (RANKL, 462-TEC) were purchased from R&D Systems
Mini-PROTEAN TGX-PAGE gels (4568046) were purchased from BioRad and PVDF membranes (IPFL0010) from Millipore

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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