SMI-4a treatment inhibits the phosphorylation of TORC1 substrates. (A) Jurkat or 6812/2 cells were incubated with 10μM SMI-4a in the absence of serum for the indicated time periods. Extracts of these cells were Western blotted with antibodies to phospho-p70 S6K (Thr389), total p70 S6K, and GAPDH. (B) Jurkat cells were treated with SMI-4a for varied periods of time and extracts were probed with antibodies to phospho–4E-BP1 (Thr37/46), total 4E-BP1, and GAPDH. (C) Identical extracts as in panel B were probed with antibodies to phospho-Akt (Ser473) and GAPDH. (D) Hematopoietic tissue was harvested from TKO mice and WT-FVB mice, and subjected to Western blots using antibodies to phospho–4E-BP1, total 4E-BP1, phospho–p70 S6K, total p70 S6K, and GAPDH as a loading control.