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Supplemental Experimental Procedures

By Takuya Yamamoto, Miki Ebisuya, Fumito Ashida, Kazuo Okamoto, Shin Yonehara and Eisuke Nishida


NIH3T3 cells by PCR and ligated into pCDNA3 containing 23 Myc tag. Cell Culture and Microinjection NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % calf serum. DB-Raf:ER cells [S1] were cultured in DMEM containing 10 % fetal bovine serum and 25 mM HEPES (pH 7.4) without phenol red. These cells were maintained in 5 % CO 2 at 37ºC. To obtain quiescent cells, cells were washed with serum-free DMEM followed by culture for 24–48 hr in DMEM containing 0.1 % serum. For FCS withdrawal, cells were washed with acid wash buffer (50 mM glycine [pH 2.8], 150 mM NaCl, 1 mg/ml polyvinylpyrrolidone), then washed with PBS and cultured in serum-free DMEM. Microinjection was performed with an IM-188 microinjection apparatus (Narishige). Samples were dissolved in injection buffer (20 mM HEPES-KOH [pH 7.4], plus 120 mM KCl). For all BrdU incorporation assays, BrdU (20 mM) was added to the culture media at the same time as growt

Year: 2013
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