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. 2002 Oct 28;159(2):217–224. doi: 10.1083/jcb.jcb.200206108

Figure 2.

Figure 2.

Inhibition of 4E-BP1, S6K1, and proliferation by rapamycin in TSC2 −/− and TSC2 +/+ MEFs. (a) Serum-starved MEFs were extracted directly or after stimulation with 200 nM insulin in the absence or presence of 20 nM rapamycin. Phosphorylation of 4E-BP1, 4E-BP1 association with 7-methyl cap–bound eIF4E, and eIF4E levels were measured by Western blot analysis as previously described (von Manteuffel et al., 1997). (b) S6K1 levels, T389 phosphorylation, and kinase activity were as in Fig. 1 a. (c) Proliferation of TSC2−/− and TSC2+/+ MEFs was determined by culturing MEFs for the indicated times in the absence or presence of 1 nM rapamycin. Each value represents the average number of cells from three independent experiments, with the cell number from each culture determined by counting three independent dishes. Filled diamonds, TSC2−/−, untreated; filled triangles, TSC2−/− with 1 nM rapamycin; filled circles, TSC2+/+, untreated; filled squares, TSC2+/+ with 1 nM rapamycin. (d) Dual phase contrast and anti-BrdU–labeled nuclei (red) images of TSC2+/+ and TSC2−/− MEFs left untreated for 4 d (UN, top) or in the presence of 1 nM rapamycin (Rapamycin, bottom). Inset shows percentage of BrdU-positive nuclei.