D6 recycling and function were impaired by WM and BFA. (A,B) CHO-K1/D6 cells were incubated at 37°C for the indicated times with medium (□), 1 μM WM (■), 35 nM BFA (▲), or both inhibitors (●), and analyzed for D6 membrane expression (A) or D6 internalization (B). (C) CHO-K1/D6 cells were incubated with 100 nM CCL3L1 for 1 hour with the indicated inhibitors, and D6 up-regulation on cell surface was evaluated as described in “D6 internalization and cell surface expression.” Data are percentages of MFI of each cell treatment over basal conditions; asterisks indicate significant differences of cells incubated with indicated inhibitor versus untreated cells calculated with Student t test and Mann-Whitney test. (D-F) CHO-K1/D6 cells were incubated at 37°C with 0.4 nM CCL4 mixed with 0.1 nM ¹²⁵I-CCL4 in the presence of indicated inhibitors. Data are the percentage of radioactivity counted (cpm) over CHO-K1 cells in the TCA-soluble (D) and TCA-insoluble (E) fractions of the supernatants and in the cell-associated fractions (F). Results shown in panels A-C (means ± SEM) are from at least 3 independent experiments performed; results of panels D through F (means ± SEM) are from triplicates of 1 representative experiment of 3 performed. Asterisks indicate significant differences of cells incubated with indicated inhibitor versus untreated cells (*P < .05; **P < .01).