Transdirect insect cell

Transdirect insect cell, an insect cell-free protein synthesis system, was obtained from Shimadzu (Kyoto, Japan). Human cDNAs (Flexi ORF clones) were purchased from Promega (Madison, WI, USA). [³H]leucine, [³H]myristic acid, and ECL prime western blotting detection reagent were from GE Healthcare (Buckinghamshire, UK). ENLIGHTNING was from PerkinElmer (Waltham, MA, USA). T7-Scribe standard RNA IVT kit was from CELLSCRIPT (Madison, WI, USA). The dye terminator cycle sequencing kit, Lipofectamine LTX and Plus reagent, MitoTracker Red CMXRos and Hoechst 33342 were from Life Technologies Corporation (Carlsbad, CA, USA). Anti-FLAG monoclonal antibody, anti-SAMM50 monoclonal antibody (WH0025813), anti-SAMM50 polyclonal antibody (HPA034537) and anti-Rabbit IgG-FITC antibody were from Sigma (St. Louis, MO, USA). Protein G-HRP conjugate was from Bio-Rad (Hercules, CA, USA). ImmunoStar LD was from Wako Pure Chemical (Osaka, Japan). X-ray film was from Eastman Kodak (Rochester, NY, USA). The other reagents used were from Wako Pure Chemical (Osaka, Japan), Daiichi Pure Chemicals (Tokyo, Japan) or Seikagaku Kogyo (Tokyo, Japan) and were of analytical or DNA grade.

In vitro translation was performed using an insect cell-free protein synthesis system, Transdirect insect cell (Shimadzu). The coding sequence of firefly luciferase RNA (from pGL3-Basic (Promega)) was fused to the murine Ucp1 5′-UTR and long 3′-UTR (amplified by PCR and verified by sequencing), and this luciferase-Ucp1 UTR was introduced into pTD1 vector at a site under the control of the T7 promoter. The mRNA encoding the cDNA was prepared using a ScriptMAX Thermo T7 Transcription Kit according to the manufacturer’s instructions. The mixture (25 μL of insect cell extract, 15 μL of reaction buffer, 1 μL of 4 mM methionine, 4 μL of mRNA (4 ng)) was divided into five equal aliquots (9 μL each) and incubated with 1 μL recombinant IMP2 proteins (20 ng) at 25 °C for 5 h. The translated luciferase polypeptide was estimated by the gain in luciferase activity using a dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions.