Fig. 1.

GRKs and PKC mediate phosphorylation of CXCR4 at Ser-346/7 following stimulation with CXCL12. (A) Sequence of CXCR4 C-terminus with Ser-324/5 and Ser-346/7 highlighted in red. (B) HEK293 cells stably expressing CXCR4 were incubated in serum-free medium for 4 hours, and then stimulated with 50 nM CXCL12 for the indicated times. Cells were washed twice with cold PBS and lysed. Phosphorylation of CXCR4 Ser-346/7 was detected by western blot using a phospho-specific antibody (pS346/7). Shown is a representative western blot using pS346/7 and tubulin antibodies from at least three independent experiments. (C) Shown is a representative western blot from at least three experiments where endogenous GRKs were knocked down using ON-TARGETplus GRK siRNAs in HEK293 cells stably expressing CXCR4 for 48 hours. (D) HEK293 cells stably expressing CXCR4 were stimulated with CXCL12 (5 nM) following knockdown of endogenous GRK2, GRK3, or GRK6 and the phosphorylation of CXCR4 at Ser-346/7 was detected by western blot. Shown is a representative blot (upper panel) and summary of the 5-minute time point (lower panel) from three independent experiments (**P < 0.01 compared with control siRNA); arbitrary units (AU). (E) HEK293 cells stably expressing CXCR4 were incubated in serum-free medium for 4 hours. Bis I (2.5 μM) was added to the cells 30 minutes prior to being stimulated with CXCL12. The phosphorylation of CXCR4 Ser-346/7 was detected by western blot. Shown is a representative blot (upper panel) and summary (lower panel) from three independent experiments (*P < 0.05; **P < 0.01 compared with control); AU. (F) GRK3 was knocked down in HEK293 cells stably expressing CXCR4 while Bis I (2.5 μM) was added 30 minutes prior to stimulation with CXCL12 (50 nM). Shown is a representative blot (upper panel) and summary from three independent experiments (**P < 0.01 compared with Bis I alone, ***P < 0.001 compared with control, +P < 0.01 compared with siGRK3 alone).