Figure 6

Effects of Btk inhibitor on arachidonate release and the phosphorylation of PLCγ2, Akt and MAP kinases. (A) Macrophages were labeled with [3H]arachidonic acid for 20 h. The cells were pretreated for 15 min with the indicated concentrations of LFM-A13 followed by stimulation with either zymosan (●) for 45 min or S.aureus (▲) for 60 min. Results are mean ± SEM (n = 3) and corrected for the release in control cultures. (B) Macrophages were pretreated for 15 min with LFM-A13 (25 μM), followed by stimulation with zymosan for 30 min. Cell lysates were immunoprecipitated with antibody against PLCγ2 followed by Western blot analysis with phosphotyrosine-specific antibody. The membrane was stripped and reprobed with antibody against PLCγ2. (C) Macrophages were pretreated for 15 min with LFM-A13 (25 μM), followed by stimulation with zymosan or S.aureus for 20 min. Equal amounts of cell lysate were run on 10% polyacrylamide gels and probed with phosphospecific antibodies against ERK and p38. The membrane was reprobed with ERK-2 antibody to verify equal loading of protein. (D) Macrophages were pretreated for 15 min with LFM- A13 (25 μM) followed by stimulation with zymosan for 30 min. Western blot analysis was performed with phosphospecific antibodies against Akt. The membrane was reprobed with ERK-2 antibody to verify equal loading of protein. Data shown in B-D are representative of three separate experiments.