Zymosan and bacteria but not LPS or peptidoglycan (PGN) induce phosphorylation of Btk. (A) Macrophages were stimulated with zymosan (zym), S.aureus (S.a.), LPS or PGN for 30 min. (B and C) Macrophages were pretreated for 15 min with either wortmannin (W, 100 nM), PP2 (5 μM), SU6656 (5 μM) or SKI-1 (5 μM), followed by stimulation with zymosan (B) or S.aureus (C) for 30 min. Equal amounts of cell lysate were run on polyacrylamide gels and probed with phosphospecific antibodies against Btk. The membrane was reprobed with ERK-2 antibody to verify equal loading of proteins. The data are representative of three separate experiments.