The BCR-SYK-AKT-FOXO1 signaling pathway is operative in tonic BCR signal-dependent DLBCL cell lines and leads to the suppression of FOXO1 activity. (A) The integrity of the BCR-SYK-AKT-FOXO1 axis was determined in BCR-dependent cell lines DHL4, DHL6, Ly1, and Ly7 after crosslinking of their BCRs, with or without R406 pretreatment. Activity of key signaling components of the pathway was assessed with phospho-specific antibodies against SYK(Y525/6), AKT(S473), and FOXO1(T24 and S256). Blots were next stripped and reprobed with antibodies against total-SYK, total-AKT, and total-FOXO1. GAPDH served as a loading control. Densitometric quantification of band intensities is provided in supplemental Table 4. (B) DLBCL cells were treated with R406 (gray line) or vehicle alone (black line) for 18 hours and subjected to single-cell phospho-flow analysis to detect tonic AKT phosphorylation at S473. Dotted line, isotype control. (C) FOXO1 phosphorylation in DLBCL cell lines treated for 18 hours with R406 or vehicle alone was analyzed by immunoblotting. Densitometric quantification of band intensities is provided in supplemental Table 5.