Btk is required for the activation of the PLCγ2- and PI3Kγ-dependent pathways. Bone marrow–derived neutrophils were plated on uncoated (unstimulated) or E-selectin–coated wells for 10 minutes, and then lysates were prepared. (A) Lysates were immunoblotted with antibodies to phosphorylated PLCγ2 (phospho-PLCγ2 [Tyr1217]), total PLCγ2 (n = 3), phosphorylated Akt (n = 3), total Akt (n = 3), phosphorylated MAPK (phospho-p38), or total p38 (n = 3). (B) Lysates were immunoprecipitated with anti-Syk, followed by immunoblotting (IB) with a general phosphotyrosine (PY; 4G10) antibody. (C) Bone marrow–derived neutrophils were plated on uncoated (unstimulated) or E-selectin–coated wells for 10 minutes, and then intracellular IP₃ levels were determined using a competitive binding assay. *P < .05 vs other groups.