Plenti cmv to puro dest

RelB (NM_006509, from OriGene, Rockville, MD, USA) was inserted in pENTR/D-TOPO (Invitrogen, NY, USA). The generated pENTR-RelB vector was recombined in the 670–1 vector (pLenti CMV/TO Puro DEST, Addgene 17293) [20 (link)] using recombination-cloning technology from Invitrogen. The eGFP was used for control cell population and has previously been described elsewhere [21 (link),22 (link)]. Lentiviruses were produced by co-transfecting vectors containing RelB or eGFP cDNA and using the ViraPower Lentiviral Packaging Mix (Invitrogen, Carlsbad, CA) in the 293FT packaging cell line. The lentiviral contructs were harvested from cell supernatants, concentrated by ultracentrifugation (20,000 rpm) and stored at −80˚C until use. For viral infection, cells were plated in 6-well plates containing 2 ml of culture media and cultured until 50-70% confluence. Infections were performed in RPMI 1640 media containing 5 μg/ml polybrene (Sigma, St. Louis, MO). Culture media was changed 16 hrs after the infection and puromycin selection was performed two days post-infection.

To increase the yield and facilitate the purification of secreted Adissp, we added a signal peptide at the N-terminus of Adissp and a 6×His tag at its C-terminus using pHL-sec vector (Addgene, #99845)^{45 (link)}. The resulting DNA fragment was cloned into pENTR1A vector (Addgene, #17398). Lentiviral construct was generated via recombination of pENTR1A-Adissp into pLenti CMV/TO Puro DEST (Addgene, #17293), and lentiviruses were produced by co-transfection along with plasmids pLP1, pLP2, and pVSVG into HEK293 cells. HEK293 cells were infected with lentiviruses and Adissp expressing stable cells were selected with puromycin. Serum-free medium from stable cells was collected, and incubated with Ni-NTA Agarose (QIAGEN, #30210) at 4 °C for 2 h. The samples were loaded into Poly-Prep chromatography columns (Bio-Rad, # 7311550). The columns were washed with washing buffer (PBS containing 5 mM imidazole (Sigma, #I5513). Adissp protein was eluted with elution buffer (PBS containing 250 mM imidazole), concentrated and then buffered in PBS. Endotoxin level (0.08 EU/mL) was measured using a commercial kit (Thermo Scientific, #88282). Purified Adissp protein was used to treat adipocytes, or intravenously inject into 3-month-old C57BL/6J male mice (1 mg/kg body weight) in a total volume of 150 μL. PBS was used as vehicle control. Adissp protein was used within 1 week after purification.

pENTR4-V5 (Addgene, Watertown, MA, USA, #17425), pLenti CMV/TO Puro Empty (Addgene #17482), and pLenti CMV/TO Puro DEST (Addgene #17293) were gifts from Eric Campeau & Paul Kaufman. pBabe-KRas-GV (Addgene #9052) was a gift from William Hahn. pCMV-VSV-G (Addgene #8454) and pCMV-dR8.2 dvpr (Addgene #8455) were gifts from Bob Weinberg. Wild-type (WT) and K-Ras-GD mutants were generated from pBabe-K-Ras by site-direct mutagenesis. Ras constructs were subcloned into pENTR4-V5 and recombined into pLenti CMV/TO Puro DEST by clonase reaction.