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3 protocols using sult1a3

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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3

Quantification of PAP and Sulfotransferase Activity

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For detection of PAP and sulfotransferase activity, the AMP-Glo system was modified such that the AMP-Glo Reagent I includes 10 mg/mL IMPAD1 (R&D Systems), a Golgi-resident PAP-specific phosphatase. This modified AMP-Glo reagent I would convert PAP in a test sample to AMP and then to ADP. The ADP formed was then converted to ATP and detected as a bioluminescent readout using the AMP Detection Solution.
PAP titration. PAP (Sigma) was titrated from 0 to 10 mM by the addition of 5 mL of the modified AMP-Glo reagent I, so the total volume was 10 mL and incubated for 1 h at room temperature. This was followed by the addition of 20 mL of AMP Detection Solution to each well and incubation for 30 min, and the light output is measured in a luminescence reader.
SULT3 activity. SULT 1A3 activity was measured using 100 mM PAPS (R&D Systems), 100 mM 1-napthol, and varying amounts of SULT 1A3 (R&D systems) in 50 mM Tris-HCl, pH 7.5, 15 mM MgCl 2 . The sulfation was carried out for 20 min at room temperature. The sulfation reaction was measured using the modified AMP-Glo system as described above.
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