Figure 1. Reticulocytes have elevated mTORC1 signaling.
(A) Diagram showing the phosphorylation sites on ribosomal protein S6 and strategy for immunoprecipitation of phosphorylated ribosomes. (B) Quantification by RNA-Seq of the abundance of each transcript in the pS6 244 immunoprecipitate (y-axis) vs the total hypothalamus (x-axis). Hba-a1 and hbb-b1 are enriched in the pS6 immunoprecipitate and labeled. (C) Total RNA was prepared from dissected hypothalami of mice that had been perfused with saline (black bars) or not perfused (white bars), and the relative abundance of each transcript was quantified by Taqman. Hba-a1 and hbb-b1 but not actin and pomc are depleted from the hypothalamus by perfusion. Values are normalized to rpl23. (D) Lysates from the mouse brain or RBCs were blotted for pS6 at the indicated sites. Neuron-specific enolase (NSE) and alpha globin are specific markers for the brain and RBCs, respectively. (E) Quantification of the relative phosphorylation of S6 at the indicated sites in the brain vs RBCs. Values are expressed as the ratio of pS6 to total S6. (F) Western blotting for pS6 235/236 in ribosomes purified from a range of mouse tissues. *p < 0.05. ***p < 0.001.
Figure 1—figure supplement 1. α-globin is not expressed in a specific population of hypothalamic neurons.
