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3 protocols using choline oxidase

1

Phospholipase D Activity Assay

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Hydroxychloroquine, PLD assay reagent amplex red 10-Acetyl-3,7-dihydroxyphenoxazine, and 2-dioctanoyl-sn-glycero-3-phosphocholine (C8-PC) were purchase from Cayman Chemical and tetracaine, methylbetacyclodextrin (MβCD), and cabbage PLD were purchased from Sigma–Aldrich. Horseradish peroxidase and choline oxidase were purchased from VWR.
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2

Phospholipase D2 Activity Assay

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Hydroxychloroquine was purchase from Cayman Chemical and tetracaine was purchased from Sigma-Aldrich. Purified PLD2 from cabbage was purchased from Sigma-Aldrich respectively. PLD assay reagent amplex red 10-Acetyl-3,7-dihydroxyphenoxazine and 2-dioctanoyl-sn-glycero-3-phosphocholine (C8-PC) were purchased from Cayman Chemical. Horseradish peroxidase and choline oxidase were purchased from VWR. Methylbetacyclodextrin (MβCD) was purchased from Sigma-Aldrich.
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3

Lipid Substrate Assay for Phospholipase D

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Bacterial (Streptomyces chromofuscus), peanut, and cabbage PLD was purchased from Enzo and sigma-aldrich respectively. Amplex Red 10-Acetyl-3,7-dihydroxyphenoxazine (Cayman chemical). Horseradish peroxidase (VWR), choline oxidase (VWR). All lipids including 1,2-dioctanoyl-sn-glycero-3-phosphocholine (C8-PC), 1-palmitoyl-2-(dipyrrometheneboron difluoride) undecanoyl-sn-glycero-3-phosphocholine (TopFluorPC), 1-(dipyrrometheneboron difluoride) undecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (TopFluorLPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC or 18:1 PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol (POPEtOH), and 1,2-dioleoyl-sn-glycero-3-phosphoethanol (DOPEtOH) were purchased from Avanti Polar Lipids (Alabaster, AL) in chloroform at stock concentrations of 10 or 25 mg/mL except C8-PC and TopFluor lipids which were purchased as powder. Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) was from Tocris Bioscience and made at a stock concentration of 1mM in ethanol. Valinomycin was also purchased from Tocris Bioscience and made at a stock concentration of 200μM in ethanol. The detergent n-dodecyl-β-D-maltoside (DDM) was purchased from Anatrace (D310S) and made at 200mM. ACMA (9-amino-6-chloro-2-methoxyacridine), was purchased from Life Technologies and made at a stock concentration of 2mM in ethanol.
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