Figure 2
Figure 2. BLyS stimulates Akt and mTOR activation. (A) Phosphorylation of Akt. Purified B cells were stimulated for the indicated times with 100 ng/mL recombinant human or murine rBLyS or 5 μg/mL of anti-Ig at 37°C in pregassed medium. Lysates were prepared from iced samples and analyzed by Western Blot, using antibodies specific for the phosphoserine (S473) and phosphothreonine (T308) residues associated with Akt activation. (B) Inhibition of BLyS induced Akt phosphorylation by the PI 3-kinase inhibitor LY294002. Akt phosphorylation on Ser 473 was followed in purified B cells treated with LY294002 or the inactive analog LY303511 for 1 hour before BLyS stimulation. (C) BLyS induced phosphorylation of the mTOR substrate 4E-BP1. Phosphorylation of E4-BP1 on Thr 37/46 was followed by unstimulated or BLyS-stimulated purified B cells. Quantitation of Western signals represent fold increases of phosphorylated 4E-BP1 relative to that found in unstimulated B cells at time zero with a correction for sample loading performed on the same Western blot stripped and reprobed for total 4E-BP1 protein. Quantitation was performed with a Molecular Dynamics Densitometer as described in “Antibodies and Western blotting.” (D) BLyS induces Akt phosphorylation, whereas incubation of B cells in media only does not. Purified B cells were analyzed over 4 hours for Akt phosphorylation on S473 in the absence and presence of 100 ng/mL of rhuBLyS. Quantitation was as described in panel C. (E) Akt phosphorylation on S473 is inhibited in purified B cells precultured with rapamycin. B cells were cultured for 24 hours with 50 nM of rapamycin and then cultured for the indicated times with media only or media with 100 ng/mL of rhuBLyS. Panel D shows the response to BLyS of B cells incubated without rapamycin. (F) Akt phosphorylation on S473 is impaired in B cells precultured in rapamycin and activated with anti-CD40 antibody. Purified B cells were cultured for 24 hours in vehicle or 50 nM of rapamycin and then stimulated with 0.5 μg/mL of anti-CD40 antibody.

BLyS stimulates Akt and mTOR activation. (A) Phosphorylation of Akt. Purified B cells were stimulated for the indicated times with 100 ng/mL recombinant human or murine rBLyS or 5 μg/mL of anti-Ig at 37°C in pregassed medium. Lysates were prepared from iced samples and analyzed by Western Blot, using antibodies specific for the phosphoserine (S473) and phosphothreonine (T308) residues associated with Akt activation. (B) Inhibition of BLyS induced Akt phosphorylation by the PI 3-kinase inhibitor LY294002. Akt phosphorylation on Ser 473 was followed in purified B cells treated with LY294002 or the inactive analog LY303511 for 1 hour before BLyS stimulation. (C) BLyS induced phosphorylation of the mTOR substrate 4E-BP1. Phosphorylation of E4-BP1 on Thr 37/46 was followed by unstimulated or BLyS-stimulated purified B cells. Quantitation of Western signals represent fold increases of phosphorylated 4E-BP1 relative to that found in unstimulated B cells at time zero with a correction for sample loading performed on the same Western blot stripped and reprobed for total 4E-BP1 protein. Quantitation was performed with a Molecular Dynamics Densitometer as described in “Antibodies and Western blotting.” (D) BLyS induces Akt phosphorylation, whereas incubation of B cells in media only does not. Purified B cells were analyzed over 4 hours for Akt phosphorylation on S473 in the absence and presence of 100 ng/mL of rhuBLyS. Quantitation was as described in panel C. (E) Akt phosphorylation on S473 is inhibited in purified B cells precultured with rapamycin. B cells were cultured for 24 hours with 50 nM of rapamycin and then cultured for the indicated times with media only or media with 100 ng/mL of rhuBLyS. Panel D shows the response to BLyS of B cells incubated without rapamycin. (F) Akt phosphorylation on S473 is impaired in B cells precultured in rapamycin and activated with anti-CD40 antibody. Purified B cells were cultured for 24 hours in vehicle or 50 nM of rapamycin and then stimulated with 0.5 μg/mL of anti-CD40 antibody.

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