Uptake of Hp91 is via clathrin-mediated endocytosis. (A) Immature human DCs were pre-treated with the phagocytosis inhibitor Latrunculin B or medium only for 30 min before incubation with biotinylated-Hp91 for 30 min. Cells were permeabilized with Cytofix/Cytoperm, stained with Streptavidin-Alexa 488, and analyzed by flow cytometry. Data are mean (±SEM) of N = 5. (B) Immature human DCs were pre-treated for 30 min with media only (Med), DMSO (D) = solvent control, the clathrin-mediated endocytosis inhibitors phenylarsine oxide (PAO) at 2 μM or chlorpromazine (CP) at 100 μM, the energy-dependent endocytosis inhibitor sodium azide (SA) at 10 mM, the caveolin-mediated endocytosis inhibitor nystatin (NY) at 20 μM, or the micropinocytosis inhibitor amiloride (AM) at 2 mM before incubation with biotinylated Hp91 (200 μg/ml) for 30 min. Cells were permeabilized, stained, and analyzed by flow cytometry as above. Results are mean (±SEM) of N = 3. **p < 0.01 compared to DMSO control; Student’s t-test.