Triolein

Rabbit polyclonal anti MIGA2 antibody (ab 122713) was purchased from Abcam. Rabbit monoclonal anti β-actin antibody was from Cell Signaling Technology (4970; RRID:AB 2223172). Almost all lipids were purchased from Avanti Polar Lipids: DOPC (850375), liver PE (840026), DGS-NTA (Ni; 709404), Liss Rhod PE (810150), Brain PI (4,5) P2 (840046), NBD-PA (810176), NBD-PS (810195), 18:1 NBD-PS (810198), NBD-PC (810133), NBD-PE (810156), NBD-cholesterol (810250), NBD-sphingomylein (810219), 16:0 PA (830855), triolein (18:1 TG; 870110). Palmitic acid (P0500) was ordered from Sigma-Aldrich. Sodium dithionite was from Sigma-Aldrich (157953). Etomoxir was purchased from MedChemExpress (HY-50202). The Hela cell line (ATCC #CCL-2; RRID: CVCL 0030) was a gift from Mals Mariappan (Yale University, New Haven, CT), C. elegans cDNA was a gift from Daniel Colon-Ramos (Yale University, New Haven, CT; Xuan et al., 2017 (link)). Plasmid for the 6xhis-PH-tethering construct (Bian et al., 2018 (link)) was a gift from Pietro De Camilli (Yale University, New Haven, CT).

Neutral lipid TLC were conducted using a protocol modified from [71 (link)]. Briefly, glass-backed silica plates (Silica gel 60 20 cm x 20 cm, Millipore) were preconditioned with hexane, air-dried, and baked at 100°C for 30 min. The TLC chamber was equilibrated with hexane: diethyl ether: acetic acid (80: 20: 3 v/v/v) for at least 2 h prior to developing. The identity of different lipid classes on the TLC plate was inferred by comparing their migration pattern to that of commercial standards run on the same plate. The following standards were used for neutral lipid analysis (5 μg each): cholesterol, palmitic acid, cholesteryl-palmitate, 1,3-diacylglycerol (1-Palmitoyl-3-stearoyl-rac-glycerol, Sigma-Aldrich), 1,2-diacylglycerol (C18:1/C18:1-DAG), and C18:1/C18:1/C18:1-triacylglycerol (triolein, Avanti Polar Lipids).

ARF1 and arf1-11 strains were grown overnight at 23 °C in YPUAD media, and diluted to 0.2 optical density (OD) in the morning. When cells reached OD 0.7, cells were treated either with cerulenin (10 µg ml⁻¹) or with the corresponding volume of DMSO. After 30 min of incubation at 23 °C, 1.5 × 10⁸ cells were collected. Then cells were shifted to 37 °C and 1.5 × 10⁸ cells were collected after 30 and 60 min of incubation. Lipids were extracted as described above. Dried lipid extracts were thawed and resuspended in 20 µl chlorophorm. Triolein (18:1 TG; Avanti Polar Lipids) was used as an internal marker. Samples were then spotted onto thin-liquid chromatography silica plates (aluminium sheets gel 60; Sigma-Aldrich), and neutral lipids separted by running in two successive chambers containing petroleum ether/diethyl ether (1:1, v/v) and petroleum ether/diethyl ether (49:1, v/v). For detection, plates were dipped into a solution containing MnCl₂, methanol and sulfuric acid (0.63 g MnCl₂^.4H₂O, 60 ml water, 60 ml methanol, 4 ml concentrated sulfuric acid) for 1 min and heated at 110 °C for 3 min. Plates were then scanned and analysed using the Fiji software.