Technical Reference #1869

1869.

Insulin-stimulated Interaction with 14-3-3 Promotes Cytoplasmic Localization of Lipin-1 in Adipocytes Miklos Peterfy; Thurl E. Harris; Naoya Fujita; and Karen Reue, University of California Los Angeles David Geffen School of Medicine, Journal of Biological Chemistry, 285(1869), (2010)
Link To Paper

Materials & Methods:
Cell Culture Transfection and Treatments—HEK293T cells were propagated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Cells were transfected with FuGENE6 transfection reagent (Roche Applied Science) according to the manufacturer’s instructions. 3T3-L1 fibroblasts were cultured and differentiated into adipocytes as described previously (15). Five days after the initiation of differentiation 3T3-L1 adipocytes were transfected by electroporation using a Nucleofector device (Amaxa Biosystems) according to the manufacturer’s instructions (Nucleofector program A-33 Nucleofector solution L). Electroporated cells were plated in collagen-coated glass-bottom cell culture dishes (MatTek Corp.) for confocal fluorescence microscopy analysis. To assess the effect of insulin signaling on lipin-1 subcellular localization adipocytes co-electroporated with lipin-1 and 14-3-3 were plated in Krebs-Ringer Hepes buffer (120 mM NaCl 5.4 mM KCl 1 mM CaCl2 0.8 mM MgCl2 20 mM HEPES pH 7.4) supplemented with 5.5 mM D-glucose and 1% fetal bovine serum. One day after plating cells were stimulated with either 100 nM insulin alone for 1 h or 100 nM insulin in the presence of 100 nM rapamycin for 1 h preceded by a 1-h preincubation with 100 nM rapamycin.

Microscopic Technique
Confocal fluorescence microscopy

Cell Type(s)
HEK293T cells