Figure 4. Effect of SB415286 on protein synthesis and cell growth.

(A) HeLa cells were incubated in EBSS containing or lacking AA for 3 h or alternatively having been depleted of AAs incubated in EBSS containing 1× AA mix (refeed) ± 100 nM insulin for 30 min. Cells were incubated with 50 μM SB415286 or 50 μg/ml cycloheximide during the AA depletion and refeed period. Puromycin (1 μM) was added 30 min prior to cell harvest. Cells were lysed and 30 μg of protein was analysed by immunoblotting using antibodies against puromycin or the proteins indicated. (B) HEK293T cells were seeded at a density of 3.5 × 10⁴ and treated 24 h post-seeding with vehicle solution and either 100 nM rapamycin or 50 μM SB415286 for up to 72 h. Cell number was quantified as described in the Materials and methods section.