Pnlip

Western blotting was performed as described previously (54 (link)). Briefly, cell lysate or fat pad lysate was prepared in radioimmunoprecipitation assay lysis buffer containing various protease inhibitors (complete and free of EDTA; Roche, Pleasanton, CA). Protein concentration (1 mg/ml) of the lysate was adjusted, boiled (10 min) in Laemmli buffer, and run on (10 mg per lane) 4 to 20% gradient polyacrylamide gels. Then, proteins were transferred from the gel to polyvinylidene difluoride membrane (Merck KGaA, Darmstadt, Germany) and blocked with 5% nonfat dry milk incubated with primary antibody of PNLIP (1:1000; Santa Cruz Biotechnology, Dallas, TX), IκB-α (1:1000; Santa Cruz Biotechnology), α-tubulin (1:500; DSHB, University of Iowa, Iowa), cytochrome c (1:1000; Santa Cruz Biotechnology), and Cox-IV (1:1000; Thermo Fisher Scientific, Waltham, MA) separately. Then, blots were probed with horseradish peroxidase–labeled corresponding secondary antibodies (1:10,000; Millipore, Billerica, MA). Band intensity was visualized by chemiluminescence using the Pierce ECL 2 Western Blotting Substrate Kit (Thermo Fisher Scientific) and quantified by densitometry as described previously (79 (link)).

Glyceryl trilinolein (GTL) and other triglycerides including triolein (GTO), tripalmitin (GTP), and mixed triglycerides 1,2-dilinoleoyl-3-palmitoyl-rac-glycerol (LLP), 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol (LOP), 1,3-dipalmitoyl-2-linoleoylglycerol (PLP), 1,2-dioleoyl-3-palmitoyl-rac-glycerol (OOP), 1,3-dioleoyl-2-palmitoylglycerol (OPO), 1,3-dipalmitoyl-2-oleoylglycerol (POP), linoleic acid (LA), palmitic acid (PA), oleic acid (OA), dimethyl sulfoxide (DMSO), and glycerol reagent were purchased from Sigma-Aldrich (St. Louis, MO). CER (Bachem AG, Bubendorf, Switzerland), IL12 (PeproTech, Rocky Hill, NJ), IL18 (R&D Systems, Minneapolis, MN), PNLIP, IκB-α, cytochrome c antibody (Santa Cruz Biotechnology, Dallas, TX), α-tubulin [Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Iowa], Cox-IV antibody, and TO-PRO-3 iodide (Thermo Fisher Scientific, Waltham, MA) were used.