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2 protocols using sult1b1

1

Sulfotransferase Enzyme Assay Protocol

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Dopamine hydrochloride, 17β-estradiol 3-sulfate sodium salt, potassium 4-nitrophenyl sulfate, pregnenolone sulfate sodium salt, DHEA 3-sulfate sodium salt dehydrate, MgCl2, and 3′-phosphate 5′-phosphosulfate lithium salt hydrate (PAPS, >99% purity) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). DHEA and 17β-estradiol were purchased from J&K Scientific LTD (Guangzhou, Guangdong, China). p-Nitrophenol and 2-aminophenol were obtained from Aladdin (Hang Zhou, Zhe Jiang, China). Dopamine 3-O-sulfate and dopamine 4-O-sulfate were purchased from TRC (Toronto, Ontario, Canada). The primary antibodies of SULT1A1, SULT1A3, SULT1B1, SULT1E1, and SULT2A1, as well as the GAPDH, were obtained from Santa Cruz Biotechnology (LA, California, USA) and Abcam (Cambridge, England, UK), respectively. TPCK (L-1-tosylamido-2-phenylethyl chloromethyl ketone)–treated trypsin was purchased from Promega (Madison, WI, USA). The recombinant human SULT isoforms (SULT1A1, SULT1A3, SULT1B1, SULT1E1 and SULT2A1) were purchased from R&D Systems (Toronto, Ontario, Canada). The solid phase extraction cartridges (C18 100 mg, 3 mL) were purchased from J.T. Baker (Philipsburg, NJ, USA). PrimeScript RT Reagent Kit and SYBR Premix Ex Taq II (TliRnaseH Plus) were purchased from Takara Bio (Shiga, Tokyo, Japan).
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2

Palbociclib Glucuronidation and Sulfation Assay

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ABT (Sigma‐Aldrich), palbociclib sulfate (TRC), uridine 5′‐diphosphoglucuronic acid, UDPGA (Sigma‐Aldrich), MgCl2 (Sigma‐Aldrich), Alamethicin (Sigma‐Aldrich), dimethyl sulfoxide, DMSO (Sigma‐Aldrich), ACN (liquid chromatography‐mass spectrometry [LC–MS] grade; Fisher Chemical), ACN (Optima LC/MS grade, Fisher Chemical) formic acid (Fisher Chemical), potassium phosphate (Sigma‐Aldrich) 5,5‐diethyl‐1,3‐diphenyl‐2‐iminobarbituric acid (“39:an”; Sigma‐Aldrich), D‐Saccharolactone (Sigma‐Aldrich), ultra‐pure water (Millipore), recombinant UGT supersomes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15; Corning), β‐estradiol (Sigma‐Aldrich), chenodeoxycholic acid (CDCA; Sigma‐Aldrich), trifluoperazine (TFP; Sigma‐Aldrich), serotonin (Sigma‐Aldrich), propofol (Sigma‐Aldrich), zidovudine (Sigma‐Aldrich), oxazepam (Sigma‐Aldrich), raloxifene (Sigma‐Aldrich), dehydroepiandrosterone (DHEA; Sigma‐Aldrich), DHEA‐sulfate (Sigma‐Aldrich), 4‐methylumbelliferone (4‐MU; Sigma‐Aldrich), palbociclib sulfate (Toronto Research Chemicals); recombinant sulfotransferases (SULT1A1, SULT1A3, SULT1B1, SULT1C2, SULT1C4, SULT1E1, SULT2A1, and SULT2B1; R&D Systems), palbociclib, and ARV‐471 were synthesized by AZ chemistry.
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