MM cells are characterized by constitutive activation of the PI3K/Akt/mTOR pathway. (A) Immunoblotting for p-mTOR at Ser2481, mTOR, Raptor, Rictor, Deptor, PTEN. and α-tubulin in MM cell lines. (B) Immunoblotting for p-rS6 Ser235/236, p-4EBP1 Thr37/46, and α-tubulin in MM cell lines. (C) In vitro Akt kinase assay and immunoblotting for p-Akt Ser473 and total Akt. For the Akt kinase assay, whole-cell lysates were immunoprecipitated with anti-Akt Ab, and then the immunoprecipitate was washed and subjected to the in vitro kinase assay according to the manufacturer's protocol. Western blot analysis was performed with anti–p-GSK3α/β. (D) BM biopsies from MM patients (n = 25) were fixed in Zanker formalin, embedded in paraffin blocks, and sectioned. Sections were then stained for p-rS6 Ser235/236 and p-GSK3α/β.