OSU-T315 impairs AKT translocation to lipid raft subdomains in plasma membrane. (A) Mec-1 cells were retro-virally transduced with pBABE-Myr-flag-AKT vector (Addgene). The total lysates were subjected to western blot to verify the myristoylated AKT expression. (B) Mec-1 expressing Myr-flag-AKT was treated with increasing concentration of OSU-T315. Total lysate was analyzed by immunoblotting. (C) Lipid raft from Mec-1 cells expressing Myr-flag-AKT was purified after vehicle or OSU-T315 (4 μM) treatment by the ultracentrifugation approach. The raft (R) and nonraft (NR) fractions were analyzed for raft-associated molecules, and Flotillin-1 serves as a lipid raft marker. (D) The 3 independent studies were quantified for AKT content in raft compartment. (E) Mec-1 cells with Myr-flag-AKT were subject to immunofluorescence staining with antibodies of α-AKT (Alexa 594) and α- Cholera toxin subunit B (CT-B) (Alexa 488). (F) The colocalization index was measured and analyzed by confocal microscope double blindly. (G) 697 cells were treated with plate-bounded α-IgM, 4 μM OSU-T315, or in combination for 1 hour, and the lipid raft fractions were extracted for analysis. Cholera toxin subunit B (CT-B) is the marker for lipid raft compartment.