Intracellular signaling of CX
CR1 and functional mutants exogenously expressed in MDA-436 cells. Western blotting analysis of MDA-436 cells that when exposed to 50 nM of human FKN failed to phosphorylate Erk1/2, as expected from the minimal expression of endogenous CX3CR1. Cells that were engineered to stably overexpress wild-type CX3CR1 (MDA-436+X) showed a significant and time-dependent Erk phosphorylation in response to FKN. Finally, cells expressing the Y14F (MDA436+Y14F) or R128N (MDA436+R128N) functional mutants of the receptor showed a negligible or lack of Erk phosphorylation, respectively; the intensity levels of Erk phosphorylation obtained from three independent experiments were evaluated by densitometry analysis and normalized using total Erk signals after membrane stripping to compensate for variations in protein loading among samples. Data are represented as mean ± S.E.M. (* P < 0.003).