GPCR signaling activates Plcβ2 and Plcβ3 in a Rac1-dependent manner. (A) Co-immunoprecipitation (co-IP) of Plcβ2 or Plcβ3 with Rac1. Bone marrow–derived WT neutrophils were left untreated or were stimulated with CXCL1 (100 ng/mL, 3 minutes, 37°C), lysed, and used for precipitation with Plcβ2 or Plcβ3 antibody and were blotted and incubated with Rac1 antibody (upper panel) or (lower panel) used for precipitation with Rac1 and then blotted and incubated with Plcβ2 or Plcβ3 antibody (n = 3 experiments for both). For densitometric analysis, see supplemental Figure 4A-B. (B-C) Number of arrested cells per square millimeter after injection of 500 ng CXCL1 in cremaster muscles of mice transplanted with retrovirally transduced Plcβ-knockout (KO) bone marrow. Chemokine-induced arrest of (B) Plcβ2-KO bone marrow nontransduced (non-transd.) or transduced with Plcβ2-complementary DNA (cDNA) or Plcβ2-ΔPH and (C) Plcβ3-KO bone marrow nontransduced or transduced with Plcβ3-cDNA or Plcβ3-ΔPH. (D) Representative western blots of WT, Gnai2^−/−, Plcβ2^−/−, or Plcβ3^−/− neutrophils, which were left untreated or stimulated with CXCL1 (100 ng/mL), lysed, and used to pull down GTP-bound active RAC1. Total RAC1 serves as loading control (n = 3 experiments). (E) Intracellular concentrations of IP₃ before and after stimulating cells with CXCL1 (100 ng/mL, 3 minutes; n = 3 experiments). #P < .05 WT vs all other groups; *P < .05, Plcβ2^−/− vs Gnai2^−/− and Rac1^−/−, and Plcβ3^−/− vs Gnai2^−/− and Rac1^−/−. (F) Bone marrow–derived Indo-1-labeled neutrophils from WT, Plcb2^−/−, Plcb3^−/−, and Rac1^−/− mice were investigated for intracellular calcium levels before and after stimulation with CXCL1. Arrow indicates CXCL1 treatment (100 ng/mL; n = 4 experiments). #P = .05. (G) Flow cytometry was used to analyze APC-labeled ICAM-1 binding in unstimulated or CXCL1 (100 ng/mL, 3 minutes) stimulated bone marrow–derived neutrophils (n = 3 experiments). #P < .05 WT vs all other groups; *P < .05 Plcβ2^−/− vs Gnai2^−/− and Rac1^−/−, and Plcβ3^−/− vs Gnai2^−/− and Rac1^−/−. (H) Percentage of clustered cells in the inflamed cremaster muscle (tumor necrosis factor-α, 2 hours; n = 4). #P < .05 WT vs all other groups. *P < .05 Plcβ2^−/− vs Rac1^−/−, and Plcβ3^−/− vs Rac1^−/−.