Figure 3. The internalization of a2NTD requires actin, microtubules and an energy source. (A) Representative histogram showing peripheral blood mononuclear cells (PBMCs) incubated with a control peptide, a2NTD conjugated to Alexa Fluor 488 (a2NTD-AF488) alone, or a2NTD-AF488 after the indicated inhibitors. Cell populations were analyzed by flow cytometry, upon gating on CD14⁺ event. (B and C) PBMCs were pre-treated with actin and microtubule inhibitors for 1 h followed by the exposure to 10 μg/mL a2NTD-AF488 for 1 h. Representative histograms for each inhibitor are shown in (B). Dashed line depicts cells incubated with a2NTD-AF488 alone, while solid lines refer to cells exposed to inhibitors plus a2NTD. (C) Bar graph values in C illustrate the percentage of a2NTD-positive cells after inhibitor treatment. (means ± SEM, n = 5, **p < 0.001, compared with a2NTD alone). (D) PBMCs were cultured in glucose-free media with or without NaN₃ and 2-deoxyglucose (to deplete ATP) or mycophenolic acid (to deplete GTP) for 2 h at 37°C, followed by the administration of a2NTD-AF488 for 1 h. Columns report mean fluorescence intensity (MFI) values (means ± SEM, n = 5, *p < 0.05, compared with a2NTD alone).