Ccds31642

pcDNA3.1 plasmids expressing full length DGAT2 (CCDS31642.1), a c.260G>A mutated version of DGAT2 and unspliced XBP1 were purchased from GenScript. Plasmids were linearized by digestion with SmaI restriction enzyme followed by removal of salts and enzymes with a column-based Nucleospin PCR cleanup system (Macherey-Nagel, 740609.50) according to the manufacturer’s protocol. The linearized plasmids served as templates for in vitro transcription using T7 RiboMAX Express Large Scale RNA Production System (Promega, P1320). RNA was purified with Nucleospin PCR cleanup system. RNA (1 µg) was incubated with recombinant IRE1α-N-His-GST fragment containing the cytosolic kinase and endoribonuclease domains (aa 465-977) (Creative Biomart, ERN1-1124H) in the presence or absence of 20 μM MKC8866 for 1 h at 37 °C in a buffer containing 40 mM HEPES pH 7.5, 2 mM MgOAc₂, 100 mM KOAc, 1 mM DTT. Products of the digestion were separated in a 1.5% agarose gel made in 1× MOPS (Fisher Scientific, BP2900-1) and visualized with DDGIT gel scanner (LI-COR).

pGenLenti plasmids expressing full length DGAT2 (CCDS31642.1) and a c.260G>A mutated version of DGAT2 were purchased from GenScript. Lentivirus was generated by co-transfecting the above plasmids with second-generation lentivirus-packaging system (Addgene, pMD2.G 12259; psPAX2, 12260; pRSV-Rev, 12253) using PEI Prime transfection reagent (Sigma-Aldrich, 919012) into HEK293T cells. Virus-containing supernatant was harvested and filtered through 0.22 μm filter (Sarstedt Filtropur 83.1826.001). MDA-MB-231 cells were transduced with this media and selected in 400 ng/ml of puromycin (Sigma-Aldrich, P8833).