Fig. 4. Oxidants and thiol antioxidants operate at different sites of CD21 shedding induction. (A) Analysis of the tyrosine phosphorylation pattern after incubation of 293 cells stably transfected with construct B (293-CD21-B cells) (left panel) and Daudi B cells (right panel) with the CD21 shedding stimuli pervanadate (200 µM) or NAC (5 mM). Cells were stimulated for 30 seconds or 2 minutes at 37°C, lysed and subjected to western blot analysis (1.6x10⁵ 293-CD21-B cells/lane; 1.3x10⁶ Daudi B cells/lane) using an anti-phosphotyrosine antibody. As loading control, both blots were stained with an anti-ß-actin antibody (lower panel). (B) Pervanadate-, but not NAC- and GSH-induced CD21 shedding is inhibited by the PKC inhibitor BIM. 293-CD21-B cells were preincubated for 1 hour with or without 10 µM BIM and subsequently incubated for 4 hours with 1 mM pervanadate, 5 mM NAC or 5 mM GSH in the presence or absence of 10 µM BIM. Cell culture supernatants were collected and sCD21 was measured by ELISA (values expressed as pg sCD21/ml/median fluorescence intensity of 293-CD21-B cells). Data are means of three independent experiments each measured in triplicate.