APRIL induces phosphorylation of mTOR and is substrates. (A) Karpas cells were stimulated for the indicated times with APRIL and analyzed for phosphorylation of mTOR, 4E-BP1, p70S6 kinase, and S6. (B) Karpas cells were cultured alone or cocultured with irradiated HS.5 cells in the presence of TACI-Fc (5 μg/mL) or control-Fc (5 μg/mL) and activation of S6 and 4EBP1 by Western blot. Data from a representative experiment are shown (n = 3). (C) Karpas cells were treated with the indicated doses of rapamycin and then stimulated with APRIL for 180 minutes, and were analyzed for mTOR phosphorylation. (D) Karpas cells were transfected with control or Akt siRNA, and then were treated with APRIL for indicated times and analyzed for 4E-BP1 phosphorylation. Expression of Akt and β-actin is shown as controls. (E) Karpas cells were treated with 100 nM or 500 nM wortmamnin or 10 μM LY294002, and then stimulated with APRIL for 180 minutes, and were analyzed for 4E-BP1 phosphorylation. (F) CD19⁺ FL B cells were treated with APRIL for the indicated time and analyzed for mTOR, 4E-BP1, p70S6 kinase, and S6 phosphorylation. Data from a representative patient are shown (n = 3). (G) DHL-6 cells were transfected with control, TACI, or BCMA siRNA (100 nM) for 72 hours, and subsequently induced with APRIL for the indicated times and analyzed for S6 phosphorylation. Densitometric values are listed below each blot.