Knockdown of GRP78 suppresses AKT signaling. (A) Representative Western blot results using BM cell lysates (n = 2 for each genotype) for detection of the indicated protein levels. (B) Quantitation of Western blot results of relative GRP78 level (n = 9 for each genotype) normalized against β-actin and the ratio of p-AKT to total AKT and p-S6K to total S6K in panel A. The ratio of one of the WT levels of GRP78, p-AKT, and p-S6K was set as 1. (C) Western blot results of lysates from HL60 cells transfected with siRNA against Grp78 (siGrp78) or control siRNA (sictrl), followed by serum starvation for 16 hours, and then stimulated with 10% serum for the indicated time (hours). (D) Quantitation of the ratio of p-AKT to total AKT in panel C. The ratio at the 0-hour time point in cells transfected with sictrl was set as 1. (E) Western blot results of lysates from HL60 cells transfected with siRNA against Grp78 (siGrp78) and control siRNA (sictrl), followed by 16-hour serum starvation, and then stimulated with 10% serum for the indicated time (hours). (F) Left: Representative immunofluorescent images of untreated (0 minutes) or serum-stimulated (3 minutes) HL60 cells transfected with either sictrl or siGrp78 and stained with anti–PI(3,4,5)P3 antibody. Scale bar represents 20 μm. Right: Quantification of relative edge fluorescence intensity of PI(3,4,5)P3 staining in serum-stimulated siRNA-transfected cells normalized to nonstimulated siRNA-transfected cells (n ≥ 30 cells per condition). The normalized relative intensity of the 3-minute stimulated sictrl cells was set as 1. Data are mean ± SE. *P < .05 (Student t test). ***P < .001 (Student t test).