SAHA did not change phospho-tyrosine proteins bound to PI3K but did directly inhibit PI3K activity. (A) Phospho-tyrosine proteins bound to PI3K. Cell lysates of Jeko1 cells cultured either with or without SAHA (5 μM, 6 hours), were immunoprecipitated with anti-PI3K p85 antibody. Precipitated proteins were fractionated on a SDS-PA gel and transferred to a membrane that was probed with an anti-phosphotyrosine antibody. (B) FOXO1a expression vector plus a vector expressing constitutive active (CA)-Akt or -PI3K were transfected into 293T. Cells were cultured in either the presence or the absence of SAHA (5 μM, 6 hours). Cellular lysate was Western blotted, and phosphorylated FOXO1a and whole FOXO1a were detected with either phospho-FOXO1a or FOXO1a antibodies. (C) PI3K in vitro assay. PI3K was precipitated using anti-p85 PI3K antibody from Jeko1 cells. The precipitated PI3K was subjected to in vitro PI3K assay using PI as substrate. Kinase assay was performed either in the presence or absence of SAHA (1 μM, 25 μM). The phosphorylated PI (PIP) was separated by chromatography. No tx, control Jeko1 cells with no treatment.