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. Author manuscript; available in PMC: 2010 May 29.
Published in final edited form as: Cell. 2009 May 14;137(5):873–886. doi: 10.1016/j.cell.2009.03.046

Figure 4. mTOR Negatively Regulates DEPTOR Protein and mRNA Expression.

Figure 4

(A) Serum starvation and stimulation regulates DEPTOR protein expression. HeLa cells were serum starved for 30 hours and stimulated with serum (10% final concentration) for the indicated times. Cell lysates were analyzed by immunoblotting for the levels of indicated proteins and phosphorylation states.

(B) DEPTOR amounts in mTORC1 and mTORC2 correlate with its expression within cells. HeLa cells were serum starved for 30 hours and stimulated with serum (10% final concentration) for the indicated times. Cell lysates and raptor and rictor immunoprecipitates were analyzed by immunoblotting for indicated proteins.

(C) DEPTOR protein expression is downregulated in cancer cell lines with constitutive mTOR signaling. Cell lines were seeded at equal density and grown in the presence or absence of serum for 30 hours and cell lysates analyzed as in (A).

(D) DEPTOR protein expression is downregulated in PTEN null or TSC2 null MEFs. Indicated MEF lines were seeded at equal density and grown in the presence or absence of serum for 30 hours and cell lysates analyzed for levels of DEPTOR, PTEN, TSC2, and raptor. An asterisk (*) indicates a non-specific band.

(E) Rapamycin treatment partially and Torin1 treatment fully rescues the serum-induced decrease in DEPTOR protein. HeLa cells were serum-deprived for 30 hours, and then stimulated with serum in the presence of 100 nM rapamycin, 250 nM Torin1, or vehicle for the specified times. Cell lysates were analyzed as in (A).

(F) Regulation of DEPTOR protein and mRNA expression. HeLa cells were serum-deprived for 30 hours, and then stimulated with serum in the presence of 100 nM rapamycin, 10 uM of MG132, 250 nM Torin1, or vehicle for the specified times. Cell lysates were analyzed by immunoblotting for the levels of indicated proteins and phosphorylation states. DEPTOR mRNA was measured by qRT-PCR and normalized to GADPH mRNA levels. Error bars indicate standard deviation for n=3.

(G) Reductions in raptor or rictor expression increase DEPTOR protein and mRNA levels. Five days after transductions with raptor or rictor shRNA-expressing lentiviruses, HeLa cells were lysed and analyzed as in (A). DEPTOR mRNA was prepared and analyzed as in (F).