Sep pak plus silica cartridges

Cells or tissue samples were homogenized in lysis buffer [20 mmol/L Tris-HCl, pH 8; 150 mmol/L NaCl, 1% Triton-X 100, protease inhibitor (cOmplete Tablets, mini EASYpack; Roche, Mannheim, Germany)]. The homogenate was centrifuged at 12,000 × g at 4 °C for 15 min to collect the supernatant. Protein concentration was determined using the DC protein assay kit (Bio-Rad Laboratories). Next, the proteins were separated by 12.5% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Target proteins were probed with HMOX1 or β-actin primary antibody (1:1000; Cell Signaling, MA, USA) at 4 °C overnight, and then incubated with HRP-conjugated anti-rabbit IgG secondary antibody (1:2000, Cell signaling) at room temperature for 1 h. Signals were visualized with the substrate Chemi-lumi One (Nacalai Tesque) using a LAS-3000 visualizer (Fujifilm, Tokyo, Japan). Protein expression level was normalized using β-actin as an internal control.
2.9. Quantification of liver siphonaxanthin accumulation by high performance liquid chromatography Siphonaxanthin was extracted from the liver tissues and subjected to HPLC analysis as previously described [16] . The lipid extracts were loaded onto Sep-Pak Plus silica cartridges (Waters, MA, USA) to remove the TAG fraction, and dissolved in methanol for HPLC analysis.
The peak of siphonaxanthin was further confirmed from its characteristic UV spectrum.