Technical Reference #1748

1748.

Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division David Michaelson; Wasif Abidi; Daniele Guardavaccaro; Mo Zhou; Ian Ahearn; Michele Pagano; Mark R. Philips, New York University School of Medicine, Journal of Cell Biology, 181(1748), (2008)
Link To Paper

Materials & Methods:
Cell culture transfection and infection: COS-1 ECV304 NIH3T3 T98G IMR-90 and MDCK cells were obtained from the American Type Culture Collection. PAE cells were obtained from M. Klagsburn (Children ’ s Hospital Boston MA). Phoenix retroviral producer cell lines were obtained from G. Nolan (Stanford University Palo Alto CA). Rac1fl ox/fl ox mouse embryonic fi broblasts were provided by V. Tybulewicz (National Institute for Medical Research London UK) and J. Kissel (Wistar Institute Philadelphia PA). Cells were grown in DME (Cellgro; Mediatech Inc.) supplemented with glucose L -glutamine sodium pyruvate 10% fetal bovine serum (for Cos-1 ECV 304 MDCK T98G IMR-90 and PAE; Cellgro) or 10% calf serum (for NIH 3T3 and Phoenix; Atlanta Biologicals) and antibiotics at 37 ° C and 5% CO 2 . For all highmagnification microscopy cells were plated transfected and imaged in the same 35-mm culture dish that incorporated a No. 1.5 glass coverslip –sealed 15-mm cut-out on the bottom (MatTek). Transfections were performed 1 d after plating at 50% confl uence using SuperFect (QIAGEN) according to the manufacturer ’ s instructions. Transiently transfected cells were analyzed 1 d after transfection. NIH 3T3 cells were infected as described previously ( Pear et al. 1993 ). In brief Phoenix cells plated on 60-mm dishes were cotransfected with the indicated MSCV-GFP plasmid and an ecotropic helper vector using Superfect with 3 ml of fresh media added 3 h after transfection. 27 and 50 h after transfection 1 ml of cleared supernatant from the Phoenix cells was added to 2 ml of fresh media on NIH 3T3 cells in the presence of 5 μ g/ml polybrene. PAE and ECV cells stably expressing GFP-Rac1 have been described previously ( Michaelson et al. 2001 ). NIH 3T3 cells stably expressing GFP-Rac1 were derived by retroviral transduction with MSCV-GFP-Rac1 and selection in puromycin. Where indicated cells were treated with 10 μ M compactin for 24 h. Puromycin compactin and polybrene were obtained from Sigma-Aldrich.

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
MDCK; COS-1; PAE cells