Phosphorylation of CCR5 induced by chemokine MIP-1β.

(A) Human monocytes were incubated in the presence or absence of MIP-1β (1 μg/mL) for 1 minute at 37°C. Cell lysates (20 μg) were electrophoresed in the presence (reducing +) or absence of 2-mercaptoethanol, transferred to IP membranes, and then immunoblotted with a polyclonal anti-CCR5 antibody. (B) CCR5-transfected human kidney embryonic 293 cells (CCR5/293) received the same treatment described in panel A. (C) Human monocytes incubated at designated times with MIP-1β (1 μg/mL) were lysed, and 200 μg cell lysates were immunoprecipitated with an antiphosphoserine antibody. The immunocomplexes were electrophoresed followed by immunoblotting with anti-CCR5 antibody. The inset shows the detection by immunoblot of CCR5 in 20 μg cell lysates prior to immunoprecipitation. (D) Densitometric measurement of the levels of phosphorylated CCR5 shown in panel C normalized to the receptor contents (inset in panel C). (E) Induction of CCR5 phosphorylation by different concentrations of MIP-1β. (F) An increased phosphorylation of CCR5 was detected by an anti-FLAG antibody in³²phosphorus-labeled 293 cells transfected with FLAG-tagged CCR5 cDNA.