The internalization assay was performed accordingly with our experimental conditions previously published [10 (link),12 (link)]. Briefly, R5-SupT1-L23 or M10 cells were exposed to CCR5 Ab Pos (1/30), and to 2 μg/mL RANTES (R&D Systems, MN, USA) as a positive control, at 37 °C for 30 min. The cells were then washed and incubated for an additional 120 min [10 (link),12 (link)]. CCR5 Ab Neg was used as a negative control [10 (link),12 (link)]. When indicated, the cells were pre-treated for 1 h with staurosporine (a pan-kinases inhibitor) (50 nM) (Sigma Aldrich, St. Louis, MO, USA). As staurosporine has been reconstituted in DMSO, the same percentage of DMSO diluted in RPMI was used as a negative control (unpublished data). The detection of cytoplasmic CCR5 was obtained by using immunofluorescence and analyzed on a Leica DMRE fluorescence microscope, according to Venuti et al., 2016 [10 (link)].
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