Subcellular Localization of GFP-Tagged Proteins

Expression of GFP-fused protein and analyses of its subcellular localization were performed essentially as described previously [23 (link)]. Briefly, expression constructs were prepared by inserting coding sequences of candidate proteins into the pAcGFP1-N1 vector (Clontech) to fuse GFP at the C-terminus of target proteins. The constructs were transfected to human HEp-2 or HEK293T cells using X-tremeGENE 9 reagent (Roche). The parental cell lines were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. After 24-h incubation, nuclear DNA and mitochondria were stained with Hoechst 33342 (Lonza) and MitoTracker Red CMXRos (Lonza), respectively. Fluorescent images were obtained using a confocal microscope, LSM710 (Carl Zeiss).