Inhibition of mTORC1 activity and protein synthesis in vivo by INK128. (A) mTORC1 activities measured by pS6 and p4EBP1 levels and (B) in vivo protein synthesis in the erythroid and non-erythroid cells in BM, spleen, and blood samples. Both Wt–Fe and Hri^−/−–Fe mice were treated with vehicle or INK128 for 6 hours. Equal numbers of nucleated cells from BM and spleen were loaded, and the exposure time for developing the western blot was the same for BM and spleen. For blood samples, equal volumes of packed cells were loaded. For measurement of in vivo protein synthesis, the entire nitrocellulose membrane was incubated with anti-puromycin antibody. Puromycin signals from the entire lane of western blots demonstrate protein synthesis activity in this particular sample, which were quantified by ImageJ software and indicated at the bottom of each lane (details available in supplemental Methods). The protein synthesis in Wt–Fe BM samples is defined as 1 for samples from BM and spleens; for Wt–Fe blood samples, it is defined as 1 for comparison of protein synthesis in blood (right panel).