LTA induces slow PMA and PLC inhibition-sensitive phosphorylation of CCR5 cytoplasmic tail. (A) Phosphorylated CCR5 band-shift observed by Western blot on total cell lysates from blood monocytes treated for 5 minutes with CCL5 (C), compared with medium (M) and LTA (L). (B) Detection of phosphorylated CCR5 in monocytes by flow cytometry on permeabilized cells with the use of a CCR5 phosphospecific (p-S349) monoclonal antibody after 15 minutes of stimulation with 100nM CCL5 or 10 μg/mL LTA [percentage of p-S349–positive cells]. SSC indicates side scatter. (C) Quantification of the average number of p-S349 CCR5-positive monocytes after 15 and 60 minutes of CCL5 (■) or LTA (□) treatment compared with untreated cells (dashed line) from ≥ 7 independent experiments; ***P < .005; ns, non significant. (D) Single slide views of Z stack confocal compressions showing untreated or CCL5- or LTA-treated monocytes (15 and 60 minutes) stained permeabilized for p-S349 CCR5. (E) Single confocal sections showing untreated or CCL5- or LTA-treated monocytes (60 minutes) incubated in the absence (Medium) or presence of 10μM PLC inhibitor (U73122) or 0.1μM phorbol esters (PMA) and stained for p-S349 CCR5 (scale bars = 10 μm).