Figure 2.

Kinetics of phosphorylation of Btk sites 1 and 2 after stimulation of alternative receptor pathways. (A) Ramos B cells were activated by crosslinking of the BCR with goat anti-human IgM for 0–30 min. (B) IL-5-dependent Y16 pro-B cells were deprived of IL-5, then stimulated 0–20 min by addition of IL-5 (lane C, 20 min, no IL-5). (C) MCP-5 mast cells were primed with anti-DNP IgE, then FcɛRI was crosslinked by addition of DNP-human serum albumin for 0–30 min. After immunoprecipitation, the phosphorylation of Btk was demonstrated by immunoblot analysis with monoclonal 4G10 antiphosphotyrosine antibody, 551PYAb, or 223PYAb. The amount of Btk protein is demonstrated by immunoblot analysis with anti-Btk N-terminal antibody.