Fig. 4.

Depletion of ROS with NAC or ebselen inhibits H₂O₂-, anti-CD40-, and SDF-1-induced Akt activation but does not inhibit anti-IgM-induced Akt activation. WEHI-231 cells were pretreated with 30 mM NAC for 2 h or with 20 μM ebselen for 1 h and then stimulated for the indicated times with a 300 μM H₂O₂, b 10 μg/ml of the 1C10 anti-CD40 monoclonal antibody, c 30 μg/ml anti-IgM or d 100 ng/ml SDF-1. When cells were pretreated with ebselen, the control cells were incubated with an equivalent volume of DMSO. Cell extracts were separated by SDS-PAGE and analyzed by immunoblotting with a phospho-specific antibody that recognizes the activated form of Akt, which is phosphorylated on serine 473 (upper panels). The blots were then stripped and reprobed with anti-Akt antibodies to show that equivalent amounts of Akt were present in each sample (lower panels). Molecular mass markers (in kDa) are shown to the left. For each panel, similar results were obtained in at least two independent experiments