Figure 6.

CXCL1-induced GTPase activity (A), intracellular Ca²⁺ mobilization (B), chemotaxis (D) and receptor internalization (C) in neutrophils from mice deficient in GRK6 (GRK6^−/−) relative to wild type (GRK6^+/+). Zymosan-elicited peritoneal neutrophils were collected from mice deficient in GRK6 (GRK6^−/−) and control littermates (GRK6^+/+). A) Membranes were prepared from zymosan-elicited peritoneal neutrophils and assayed for time-dependent CXCL1-stimulated Pi released. Results shown are representative of one of two experiments performed in triplicate. *P<0.05; **P<0.01, Student’s t test. B) Cells (3 × 10⁶ cells) were Indo-1–loaded pretreated with or without CXCL1 (100 nM) and stimulated with 10 nM CXCL1. The data shown are representative of at least 3 traces. C) Cells (0.5×10⁶ cells) were treated with CXCL1 (100 nM) for different period of time and assayed for ¹²⁵I-CXCL1 binding. Data are represented as percentage of total ¹²⁵I-CXCL8 bound to control (untreated) cells. **P<0.01, Student’s t test. D) Cells were incubated with calcein AM ionophore for 30 min and resuspended (1.0 × 10⁵/20ml) in RPMI without phenol red. Different concentrations of CXCL8 were loaded in a neuroprobe 96-well plate. The cells were added to the top of the filter and incubated for 2 h at 37 °C. After incubation, the top of the filter was washed five times with medium and fluorescence intensity of the bottom well-plate was measured in a Perkin Elmer fluorescence microplate reader. The experiment was repeated 3 times in triplicate. **P<0.01; *** P < 0.001, Student’s t test.