Oligo

Lentiviral plasmids used the pHRSIN backbone 64 and the packaging plasmids pMD.G (lentiviral VSVG) and pMD.GagPol (lentiviral Gag/Pol). IMAGE cDNA clones for AUP1 (IMAGE ID: 5578910) and MARCH6 (IMAGE ID: 40148713) (Source Bioscience) were cloned into pHRSIN.pSFFV.pGK.Puro with an N‐terminal or C‐terminal HA tag (HA‐AUP1 and MARCH6‐HA). pDEST17‐UbE2J2 was a gift from Wade Harper (Addgene plasmid #15794) and cloned using Gibson Assembly^® to create pHRSIN‐pSFFV‐FLAG‐UBE2J2‐pGK‐Blasto. pMXs‐3XHA‐EGFP‐OMP25 was a gift from David Sabatini (Addgene plasmid # 83356). pHRSIN.pGK.Puro HA‐UBE2G2 was cloned from pcDNA3 Ube2G2‐myc (gift from John Christianson). pHRSIN.pGK.Puro TRC8‐HA and pHRSIN.pGK.Puro TRC8ΔRING‐HA were cloned from pcDNA6 TRC8‐HA 19. The catalytically inactive mutants (MARCH6 C9A and UBE2G2 C89A) were generated using site‐directed mutagenesis. The GFP‐CL1 (GFPu) construct was a gift from Ron Kopito. The CL1 sequence was amplified and cloned at C‐terminus of mCherry (pHRSIN‐mCherry PGK‐Puro). CL1 mutations were introduced by oligos (Sigma) and cloned into pHRSIN‐mCherry lentivirus vector. The SPP‐expressing lentiviral plasmids (pHRSIN SPP‐myc) and the ubiquitin lentiviral constructs, expressing a His‐ubiquitin/GFP (pHRSIN His‐Ub‐GFP), which result in co‐translational cleavage of GFP from ubiquitin, have been described previously 27, 38.

For making mutants for the study, site-directed mutagenesis was performed using the kit and guidelines given in the QuickChange™ Site-Directed Mutagenesis Kit from Stratagene. Oligos were procured from Sigma-Aldrich.

Anti-FLAG antibody (Sigma-Aldrich, A8592), anti-GFP antibody (Roche, 11,814,460,001), anti-GAPDH antibody (Ambion, AM4300), anti-H4 (Millipore, 07-108) and oligos (Sigma-Aldrich) were used.

MSAP was used for the DNA methylation profiling of barley plants according to the method of Rodríguez López et al. (2012) (link). To ensure marker reproducibility, DNA samples were analyzed in two technical replicates. Thus, samples were digested using a methylation insensitive restriction enzyme EcoRI in combination with either HpaII or MspI (isoschizomers), which show differential sensitivity to cytosine methylation at CCGG positions. Digested DNA fragments were ligated to adapters (Table 1) with one end cohesive with restriction products generated by EcoRI or HpaII/MspI. Digestion and ligation reactions were performed in a single solution of 11 µl comprising: 1.1 µl T4 ligase buffer; 0.1 µl HpaII; 0.05 µl MspI; 0.25 µl EcoRI; 0.05 µl T4 ligase; 0.55 µl BSA ; 1.1 µl NaCl ; 1 µl Adapter EcoRI; 1 µl Adapter HpaII/MspI; 5.5 µl DNA sample and 0.3 µl pure water. Enzymes and buffer were acquired from New England Biolabs, Australia (NEB) and oligos were produced at Sigma-Aldrich, Australia. The solution was incubated for 2 h at 37°C, then enzymes were inactivated at 65°C for 10 min.

BMDMs collected from lipin-1^mEnzyKO, lipin-1^mKO, and littermate control mice were seeded at a density of 150,000 cells per well on XF24 cell culture microplates and allowed to incubate for 4 h. Experiments were conducted in XF assay medium containing 25 mM glucose, 2 mM l-glutamine, and 1 mM sodium pyruvate and analyzed using a Seahorse XFe 24 extracellular flux analyzer (Agilent Technologies). Where indicated, the following were injected: ATP-synthesis inhibitor Oligomycin (Oligo; 1 μM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (2 μM) to uncouple ATP synthesis, rotenone (100 nM) to block complex I, and antimycin A (1 μM) (Sigma-Aldrich) to block complex III. Oxygen consumption rate (OCR) was analyzed using Wave Desktop software (Agilent Technologies).