(A) LARP1 knockdown reduces S6K1, S6, and 4EBP1, but not Akt, phosphorylation. LARP1 was knocked-downed with two distinct shRNA constructs (UTR-2 and 1–3), and levels of S6K1, S6, 4EBP1, Akt, and mTOR phosphorylation were monitored under the indicated conditions. (B–C) The effects of LARP1 knockdown on growth factor/amino acid-induced S6K1, S6, 4EBP1, Akt, and mTOR phosphorylation in LnCap and HEK293E (insulin sensitive) cells. (D) The effect of LARP1 knockdown on mTORC1 kinase activity. LARP1 was knock-downed in HEK293T cells, and mTORC1 kinase activity was measured in vitro using recombinant GST-4EBP1 as a substrate. (E) The effect of LARP1 knockdown on growth factor/amino acid-induced mTORC1 localization on lysosomes. HEK203T cells were starved with HBSS for 1 hr and stimulated with DMEM containing 10% FBS. mTOR localizes on lysosomes in response to growth factor/amino acid stimulation under LARP1 knockdown conditions.