Figure 3

PP2 inhibits the phosphorylation of Tyr396 in the activation loop of the kinase domain of Lyn or equivalent position in other SFKs, leading to a disruption of early BCR signaling events. (Panel A) A number of B lymphoma cells were treated with 10 μM PP2 for 1 hr and then cell lysates were prepared for Western blotting. The blot was first probed with p-Src (Y416) antibody, then stripped and re-probed for β-actin. 'Bc' stands for primary B cells. (B) SudHL-4 lymphoma cells were treated with or without 10 μM of PP2 for the indicated time points and cell lysates were prepared for Western blotting. The blot was first probed with p-Lyn (Y507) antibody, then stripped and re-probed for total Lyn. The ODs for p-Lyn were normalized to total Lyn and depicted as a fold change over 1 hr without PP2. (C) SFK inhibition blocks constitutive phosphorylation of Igα in B lymphoma cells. SudHL-4 cells were treated with or without 10 μM PP2 for 1 hr. Igα was immunoprecipitated from equal amount of cell lysates from both groups and analyzed by Western blotting for p-Tyr. The lysates were also probed for Igα as a loading control. The ODs for p-Tyr were normalized to Igα and depicted as a fold change over no PP2 added. (D) SFK inhibition blocks constitutive phosphorylation of CD19 in B lymphoma cells. BKS-2 and SudHL-4 cells were treated as indicated for 4 hr. For a positive control, SudHL-4 cells were treated with 20 μg/ml anti-Ig for 15 min. The cell lysates were analyzed by Western blotting with pCD19 antibody. Then the blots were stripped and re-probed with anti-β-actin antibody. The ODs for pCD19 were normalized to the corresponding β-actin bands and then for each time point, depicted as a fold change over medium only for each panel.