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. 2003 Jun 1;17(11):1352–1365. doi: 10.1101/gad.1089403

Figure 4.

Figure 4.

mTOR activity in WT and DKO MEFs. (A) Phosphorylation and protein levels were determined by immunoblotting. (Lanes 1,2) Proliferating (P) cells (passage 4) were plated in 10% FCS and analyzed 24 h after plating. (Lanes 3,6) Cells were plated in 10% FCS for 24 h, then deprived of serum and analyzed 24 h later. (Lanes 4,5,7,8) After 24 h of serum deprivation, cells were stimulated with 20% FCS for 30 and 60 min, respectively. Protein extracts from proliferating, serum-deprived, or serum-stimulated cells were subjected to immunoblotting using anti-phospho-Ser 473 of Akt (Akt-p), anti-pan-Akt (Akt-total), anti-phospho-Ser 65 of 4E-BP1 (4E-BP1-p), anti-4E-BP1 (4E-BP1-total), anti-phospho-Thr 389 of S6K1 (S6K1-p), anti-S6K1 (anti-S6K1), or anti-β-actin (loading control). (B, top panel) TSC2 phosphorylation was determined by immunoblotting using anti-phospho-Thr 1452 of TSC2. (Bottom panel) Immunoprecipitation with anti-TSC2 followed by immunoblotting with anti-Akt-p-S/T substrate. ns, nonspecific band. (Lanes 1,4) Cells were plated in 10% FCS for 24 h and then deprived of serum and analyzed 24 h later. (Lanes 2,3,5,6) After 24 h of serum deprivation, cells were stimulated with 20% FCS for 30 and 60 min, prior to analysis.