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. 2016 Jun 1;6:26812. doi: 10.1038/srep26812

Figure 6. Transactivation of CXCR4 by SCF/c-kit signaling is mediated through GRK6.

Figure 6

(A) CSCs were transfected with either GRK2 or GRK6 siRNAs. Then, cells were stimulated with 100 ng/ml of SCF for 10 minutes. The efficiency of transfection and the phosphorylation of CXCR4-serine 339, p38 MAPK and ERK1/2 were analysed using western blotting (left panel), and the data were quantified in the right panel (±SEM, three independent experiments). (B) After transfection with GRK2 or GRK6 siRNA, the chemotaxis of SCF (100 ng/ml) on CSCs was measured by the Transwell migration assay. Data represent the mean ± SEM of three independent experiments performed in duplicate. *p < 0.05; **p < 0.01; ***p < 0.001. (C,D) Overexpression of GRK6 with the pretreatment of c-kit blocking antibody in CSCs. CXCR4-serine 339, p38 and ERK1/2 phosphorylation and also migration in cells overexpressing GRK6 were enhanced relative to that in control cells, but after blocking c-kit, they were reduced even upon overexpression of GRK6. (C) Data represent the mean±SEM of three independent experiments performed in duplicate. *,#,▿p < 0.05 (c-kit ab+GRK6 plamid+SCF vs GRK6 plasmid+SCF). (D) Data represent the mean ± SEM of three independent experiments performed in duplicate. *p < 0.05; **p < 0.01; ***p < 0.001. Full immunoblots for the cropped images presented here are provided in the Supplementary Information.