Figure 2

Syk kinase activation and phosphorylation in response to BCR engagement (A) The immune complex kinase assay (ICKA) was used to determine Syk activity in BCL₁.3B3 cells after incubation with rabbit anti-IgM antibody (0 – 20 μg; lanes 1 – 5) or control rabbit anti-ova antibody (20 μg; lane 6) for 0.5 minutes. Samples were lysed in an NP-40 lysis buffer, Syk was immunoprecipitated with rabbit anti-Syk-coated protein A agarose beads, and incubated with γ-³²P ATP. The immunoprecipitates were resolved by SDS-PAGE and visualized using a phosphorimager. As immunoprecipitation controls, BCL₁.3B3 cells were lysed and samples immunoprecipitated with rabbit α-ova-coated beads (lane 7) or with uncoated beads (lane 8). The arrowhead marks the migration position of Syk. (B) The kinetics of Syk activation was determined by the (ICKA). Syk was immunoprecipitated from BCL₁.3B3 cells incubated with 15 μg of anti-IgM for 0–10 minutes (lanes 1–5) and analyzed as described above. As an immunoprecipitation control, cells were stimulated, lysed, and incubated with anti-ova-coated beads (lane 6). The arrowhead marks the migration position of Syk. (C) Phosphotyrosine levels were determined by anti-phosphotyrosine (anti-PY) immunoblotting. BCL₁.3B3 cells were unstimulated (lane 1) or stimulated with 15 μg of anti-IgM (lanes 2 and 3) or anti-ova (lanes 4 and 5) for 0, 0.5, or 5 minutes as indicated. After the cells were lysed, Syk was immunoprecipitated, resolved by SDS-PAGE under non-reducing conditions, transferred to nitrocellulose, and probed with anti-PY antibody (top panel). The blot was stripped and reprobed with an anti-Syk antibody (bottom panel). The arrowhead marks the migration position of Syk.