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. 2003 Dec;23(23):8862–8877. doi: 10.1128/MCB.23.23.8862-8877.2003

FIG. 4.

FIG. 4.

mTOR regulates UBF expression. (A) NIH 3T3 cells were made quiescent, pretreated with 20 nM rapamycin or vehicle (ethanol) for 30 min, stimulated with serum (by growing in DMEM containing 10% FBS for 24 h), and harvested. Equal amounts of protein were analyzed for UBF expression by Western blotting using polyclonal antibodies that recognize both forms of UBF (UBF1 and UBF2) (α-UBF) and an anti-actin antibody (α-Actin) to verify loading. Samples from duplicate cultures are shown. (B) NIH 3T3 cells were transfected with pSMECAT (0.45 μg) and where indicated with increasing amounts (0.9 and 1.8 μg) of a vector driving expression of UBF antisense RNA (pCMV5-UBFas) or the empty vector (pCMV5) and then transferred to DMEM containing 0.5% BSA. After 24 h of serum starvation, cells were pretreated with rapamycin (20 nM) or vehicle (ethanol) for 30 min before stimulation with DMEM containing 10% FBS for a further 24 h. The cells were harvested and assayed for CAT activity. (C) NIH 3T3 cells growing exponentially in the presence of DMEM containing 10% FBS were treated with rapamycin (20 nM) for 30 min or 3 h, harvested, and assayed by Western blotting for UBF expression as described above for panel A. (D and E) NIH 3T3 cells were transfected with pSMECAT (0.45 μg) and where indicated, with increasing amounts of a construct driving the expression of an epitope-tagged version of UBF1 (FLAG-tagged UBF-1 [FLAG-UBF1]) (0.9 and 1.8 μg) or the empty vector (pCDNA3) and then transferred to DMEM containing 0.5% BSA. After 24 h of serum starvation, cells were pretreated with rapamycin (20 nM) or vehicle (ethanol) for 30 min before stimulation with DMEM containing 10% FBS for a further 24 h as indicated. The cells were then harvested and assayed for CAT activity (D) or expression of recombinant UBF using an anti-FLAG antibody; actin was used as a loading control (E). Values that are significantly different from the control values (*, P < 0.05; **, P < 0.01) and values that are significantly different (P < 0.05) from the values for serum-treated cells (n = 5) (#) are indicated. Abbreviations: Ctl, control (serum starved); S, 10% FBS; S+Rapa, 10% FBS plus 20 nM rapamycin.