BCR crosslinking leads to PI3K-dependent down-regulation of gene expression accompanied by phosphorylation and nuclear export of FOXO1. (A) Independent RNA samples derived from separate preparations of murine B cells were analyzed by microarray (Affymetrix) or Q-PCR for expression of cyclin G2 (left) or pRb2/p130 (right). Relative expression in microarray data refers to the mean hybridization signal from replicate experiments after normalization of overall chip signals. For Q-PCR data, relative expression was calculated from standard curves and normalized to β-actin mRNA levels. Q-PCR experiments used RNA from Balb/c B cells stimulated with anti-IgM in the presence of a maximal inhibitory concentration (10 μM) of the PI3K inhibitor LY294002 (LY), whereas a suboptimal LY concentration (3 μM) was used in the microarray experiment.¹⁷  Data are plotted as percentage of control, defined as the expression level in fresh unstimulated B cells. Relative expression at T = 0 minute (▪), T = 2 hours (▦) , and T = 2 hours + LY (□) is shown. (B) Whole-cell lysates were prepared from resting primary B cells and either Cos7 cells transfected with FLAG-tagged FOXO1 and HA-FOXO3a or untransfected. These samples were then resolved by SDS-PAGE and immunoblotted for total FOXO1 and FOXO3a. The slower mobility of the transfected samples is the result of the epitope tags. Anti–β-actin was used to determine equal loading. (C) B cells were either lysed immediately (T = 0 minute) or stimulated with anti-IgM for the indicated times in the presence or absence of LY (15-minute pretreatment). Whole-cell lysates were resolved by SDS-PAGE and immunoblotted for phospho-FOXO1 (Ser256) and total FOXO1. The blot was also probed for β-actin to verify equivalent loading. (D) B cells were either harvested immediately (T = 0 and T = 0 + LY) or stimulated for 30 minutes (T = 30 and T = 30 + LY) with anti-IgM in the presence or absence of 10 μM LY294002. Nuclear and cytoplasmic lysates were loaded on the basis of cell equivalents and resolved by SDS-PAGE, followed by immunoblotting for phospho-FOXO1 (Ser256) and total FOXO1. (E) (Left) Expression levels of FOXO1 and phospho-FOXO1 (pFOXO1) in panel D were quantitated by using NIH Image 1.61. Expression is shown in arbitrary units. (Right) Ratio of cytoplasmic to nuclear FOXO1 expression. Left panel shows expression of phospho-FOXO1 in the cytoplasm. For immunoblots, similar results were obtained in 2 to 4 replicate experiments.