, After an overnight incubation at 4 1C with gentle shaking, protein A-sepharose beads were centrifuged and washed three times with ice-cold-modified IPP50 buffer. The pellet was separated in two fractions. One fraction was analyzed for RNase L protein content by western blot as described below. RNAs were extracted from the other fraction by adding TRIzol (1 ml) to the beads and following the manufacturer instructions (Invitrogen). Glycogen (10 mg) was added to ease RNA precipitation with isopropanol. cDNA was generated from total extracted RNA by RT, Briefly, confluent WT-MEFs and RNase L À/À -MEFs were lysed with IPP50 buffer (10 mM Tris-HCl

. Mrna-stability and L. Wt-mefs-and-rnase, mg/ml) for 0, 1, 2, 4 or 6 h. Then RNAs were extracted and analyzed after RT by semi-quantitative PCR as described above. Following agarose gel electrophoresis, pictures of the gels were saved as western blotting (Odyssey Infrared Imaging System LI-COR Biosciences, ScienceTec, Courtaboeuf, France) for 1 h and incubated with a rabbit polyclonal anti-RLI antibody (Novus Biological, Interchim, Montluçon, France), or a mouse monoclonal anti-Hu-RNase L (Abcam) or a rabbit polyclonal anti-mouse RNase L (Santa Cruz), or a rabbit polyclonal anti-phospho-Akt (Ser 473 ) or a rabbit polyclonal anti-Akt (both from Cell Signaling), or a mouse monoclonal anti-a-tubulin (Sigma-Aldrich) in the same buffer overnight at 4 1C. Membranes were washed in PBS supplemented with 0.05% (V/V) Tween 20 then with PBS alone and incubated for 45 min at room temperature with a donkey anti-rabbit or anti-mouse conjugated to IRDye 700DX or IRDye800, GM and

V. Rnase-l-À/À--mefs, L. -rnase, and . Hu-rnase-l-À/À--mefs, 37 After differentiation, cells were incubated 10 min with or without insulin (1 nmol) then medium was changed to glucose-free DMEM and 6-NBDG (300 mM) was then added to all cells for 45 min at 37 1C. Cells were thoroughly washed to remove all exogenous fluorescent glucose analog, and fluorescence intensity of 6-NBDG was measured at 540 nm wavelength (465 nm excitation wavelength), Glucose uptake. A fluorescent glucose analog, 6-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxy-glucose (6-NBDG, Molecular Probes

, ROS production in WT-MEFs and RNase L À/À -MEFs. ROS level was measured using the nitroblue tetrazolium (NBT) assay

. Ectopic-expression-of-hu-rnase-l-in-rnase-l-À/À--mefs, The coding sequence of Hu-RNase L cDNA was subcloned in pcDNA3neo (Invitrogen) by the standard procedures. 39 RNase L-pcDNA3neo or the empty vector pcDNA3neo were transfected into RNase L À/À -MEFs with JetPEI (Qbiogene, Illkirch, France) following the manufacturer instructions. As observed previously, the yield of transfection was low, 40 so transfectants were selected by culturing cells in the presence of 1 mg of G418 (Gibco-BRL, Fisher Scientific) per ml of cell culture medium. The polyclonal cell population expressing the transfected Hu-RNase L cDNA was named Hu-RNase L À/À -MEFs; the polyclonal cell population expressing the

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