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
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