Effect of dynasore on CCL5- and CXCL12-induced CCR5 and CXCR4 internalization, respectively. (A-B) Flow cytometry analysis of cell-surface CCR5 (A) or CXCR4 (B) using 1/85a Alexa Fluor 647–conjugated anti–human CCR5 (A) or PE-conjugated anti–human CXCR4 (B) in THP-1 cells expressing mock, Flag-UL33-YFP or Flag-UL78-YFP and pretreated or not with dynasore (100μM) for 1 hour before 15 minutes CCL5 (100nM) or CXCL12 (10nM) stimulation. The proportion of surface CCR5 (A) and CXCR4 (B) was calculated by comparing the signal in nonpermeabilized and permeabilized cells in 3 independent experiments. Values were normalized to the levels seen in each group in the absence of ligand and dynasore. (C-D) Flow cytometry analysis of cell-surface UL33 and UL78 using rabbit anti-Flag as primary antibody and rabbit-Cy5 as secondary antibody in THP-1 cells expressing mock, Flag-UL33-YFP (C) or Flag-UL78-YFP (D) after CCL5 (100nM) or CXCL12 (10nM) stimulation for 15 minutes pretreated or not with dynasore (100μM) for 1 hour. The proportion of surface UL33 or UL78 was calculated by comparing the signal in nonpermeabilized and permeabilized cells in 3 independent experiments. Mock = pcDNA3 expression vector. Statistical differences compared with unstimulated cells were assessed using the Student t test (*P < .05; **P < .01).