Phosphor imaging screening

Erythrocyte Sphk1 activity was measured using previously described methods^{8 (link)}. Briefly, RBCs were lysed in a pH7.4 Tris-HCl buffer containing 1 mM EDTA, 1 mM β-Mercaptoethanol, 0.3% Triton X-100, 50% glycerol and protease and phosphatase inhibitors. Then, the lysates were assayed using 250 µM D-erythro-sphingosine in bovine serum albumin (0.4%) and [γ-³²P]ATP (10 μCi, 20 mM) containing 200 mM MgCl₂. Lipids were extracted and then resolved by TLC on silica gel G60 with 1-butanol/methanol/acetic acid/water (80:20:10:20, v/v). The plates were then exposed to phosphor-imaging screening (Bio-Rad) and scanned for radioactive signals as indications of the amount of S1³²P synthesized.

Erythrocyte LPCAT activity was determined by measuring the formation of [¹⁴C] PC from lysoPC and [¹⁴C] acyl-CoA26 (link). All reactions were performed in 100 mM Tris-HCl, pH 7.4 containing 1 μM CaCl2, 0.015% Tween-20, 200 μM lysoPC (Avanti Polar Lipids), and 20 μM [1-¹⁴C] oleoyl-CoA (0.01 μCi) (Perkin Elmer) at 37 °C with isolated erythrocyte membrane protein in a total volume of 200 μl. The reaction was terminated by adding 800 μl of chloroform/methanol (2:1, vol/vol) to the incubation mixture. Then lipids were extracted and then resolved by Thin-layer chromatograph (TLC) silica plates with chloroform/methanol/acetic acid/0.9% NaCl (100:50:16:5, vol/vol). TLC plates were then exposed to phosphor-imaging screening (Bio-Rad) and scanned for radioactive signals as indications of the formation of PC. A part of membrane protein was used to detect LPCAT1 protein expression by Western blot. Approximately 20 μg membrane protein was run on 10% SDS-PAGE gels. After protein was transferred to PVDF membrane, the membrane was blocked with 5% nonfat milk and incubated with anti-LPCAT1 antibody (ab94903, Abcam) and Donkey anti-Rabbit secondary antibody (Santa Cruz Biotechnology Inc.), respectively.

Erythrocyte Sphk1 activity was measured using previously described methods52 (link)53 (link) with a few modifications46 (link). Briefly, RBCs were lysed in a pH 7.4 Tris-HCl buffer containing 1 mM EDTA, 1 mM β-mercaptoethanol, 0.3% Triton X-100, 50% glycerol and protease and phosphatase inhibitors. Then, the lysates were assayed using 250 μmol l⁻¹D-erythro-sphingosine in bovine serum albumin (0.4%) and [γ-³²P]ATP (10 μCi, 20 mmol l⁻¹) containing 200 mmol l⁻¹ MgCl₂. Lipids were extracted and then resolved by TLC on silica gel G60 with 1-butanol/methanol/acetic acid/water (80:20:10:20, v/v). The plates were then exposed to phosphor-imaging screening (Bio-Rad) and scanned for radioactive signals as indications of the amount of S1³²P synthesized.