Igepal ca 630

Nb leaves were ground in liquid nitrogen and resuspended in extraction buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 5 mM DTT, 2.5 mM NaF, 1.5 mM Na₃VO₄, 1x Complete EDTA free protease inhibitor cocktail (Roche), and 2% (v/v) IGEPAL CA-630 (MP Biomedicals)). In the immunoprecipitation assay, the supernatant was incubated with anti-HA magnetic beads (Miltenyi Biotec). The beads were washed four times with the extraction buffer and resuspended in an equal volume of 2× SDS sample buffer. Co-immunoprecipitated proteins were analyzed by immunoblots. To purify the microsomal fraction, tissue was homogenized on ice with extraction buffer 1 (20 mM Tris-HCl pH7.5, 0.33 M sucrose, 1 mM EDTA, protease inhibitor cocktail (Roche), and 0.1% 2-Me-OH). Crude protein extracts were centrifuged at 2000 x g to remove cell debris, and the supernatants were then centrifuged at 82,300 x g for 1 h to pellet microsomal fractions. Supernatants were used as soluble proteins. Pellets were suspended in extraction buffer 2 (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 5 mM DTT, 2.5 mM NaF, 1.5 mM Na₃VO₄, 1x Complete EDTA free protease inhibitor cocktail (Roche), and 1% (v/v) IGEPAL CA-630 (MP Biomedicals)) and used as microsomal proteins. α-PIP1 (Agrisera) was used as a PM-localized protein marker.

Blood was collected from the second cohort of animals during dissection and stored in 10% EDTA on ice. Within two hours, the blood was centrifuged at 3000 g for 10 minutes at 4°C. The supernatant (plasma) was collected and stored at -30°C until analysis. Frozen liver powder was weighed out and homogenized in ice-cold lysis buffer (5% Igepal CA-630, MP Biomedicals, Ohio, USA) with addition of Protease Inhibitor Cocktail (Sigma Life Science, Missouri, USA). Homogenization was completed using the Qiagen TissueLyser (Ontario, Canada). Samples were then centrifuged at 16000 g for 10 minutes at 4°C, supernatants were collected. Protein assays were completed (Pierce^™ BCA Protein Assay Kit, Thermo Scientific, Illinois, USA) to determine protein content for normalization. Triglyceride assays were completed to determine hepatic and plasma triglyceride content (Triglyceride Colorimetric Assay Kit, Cayman Chemical Company, Michigan, USA). NP-40 from the kit was replaced with a chemically indistinguishable substitute, 5% Igepal CA-630 (MP Biomedicals, Ohio, USA).

Reagents and their sources were as follows: RPMI 1640 medium, Oligo(dT)_12–18 Primer, SuperScript® III Reverse Transcriptase, SYBR® Green PCR Master Mix, pENTR™/D-TOPO® vector, Gateway® pT-Rex™-DEST30 vector, pT-Rex/GW-30/lacZ vector, pcDNA™6/TR vector, Lipofectamine® 2000 Transfection Reagent and blasticidin S HCl (Thermo Fisher Scientific, Waltham, MA, USA); docosatetraenoic acid, eicosapentaenoic acid (EPA) (NU-CHEK PREP, Inc.; Elysian, MN, USA); oligopeptides (Hokkaido System Science, Sapporo, Hokkaido, Japan); Tris, dithiothreitol, sodium orthovanadate, phenylmethanesulfonyl fluoride, and doxycycline hyclate (Sigma-Aldrich, St. Louis, MO, USA); sodium chloride (Nacalai Tesque, Kyoto, Japan); EDTA, sodium deoxycholate, sodium fluoride, sodium dodecyl sulfate (SDS), 4% paraformaldehyde and crystal violet (Wako, Osaka, Japan); IGEPAL CA-630 (MP Biomedicals, Santa Ana, CA, USA); protease inhibitor cocktail tablet (Complete, Mini, EDTA-free), geneticin (G418) (Roche Diagnostics GmbH, Mannheim, Germany); and SacI, XhoI (Takara Bio Inc., Otsu, Shiga, Japan).