Effect of RTX on BCR-induced Lyn activation. (A) DOHH2 were preincubated with F(ab′)2 RTX (10 μg/mL, 16 hours) and then BCR was stimulated for the indicated times. Lyn activation was evaluated by an anti–phospho-Src antibody (Y416) revealing p53 and p56 Lyn isoforms. This result is representative of 3 independent experiments. (B) DOHH2 were preincubated with F(ab′)2 RTX (10 μg/mL, 16 hours) and stimulated with anti-IgG antibody (10 μg/mL, 5 minutes). Then, Lyn-dependent Syk phosphorylation was evaluated by flow cytometry using phycoerythrin-conjugated phospho-Syk (Y348 or Y352) antibodies. Nonstimulated and stimulated cells are represented by gray and black histograms, respectively, and are representative of 3 independent experiments. (C) Syk/BCR interaction was evaluated using DOHH2 preincubated with F(ab′)2 RTX (10 μg/mL, 16 hours) and stimulated or not with 10 μg/mL of anti-IgG antibody during 1 minute. BCR Igα associated with Syk was revealed by Western blot analysis. (D) SHP-1 was inhibited by 0.1mM sodium orthovanadate (OV) during 1 hour or depleted with SHP-1 siRNA. Then, FL cells were preincubated with RTX (16 hours, 10 μg/mL) and treated with anti-IgG (10 μg/mL, 5 minutes). Results represent Syk phosphorylation (Y525) expressed in MFI and are the mean of 3 independent experiments ± SD. (Inset) SHP-1 expression analyzed by Western blot in FL cell lines transfected by control or SHP-1 siRNA.