Fig. 1. Knockdown of dynamins inhibits the internalization of gp120 in fibroblasts.

a Fibroblasts were grown for two days in the absence (−) or presence (+) of tamoxifen. Cell lysates were then prepared for Western blot analysis with an antibody against dynamin I and II. The blot was reprobed with β-actin as a loading control. b–d Representative immunofluorescent images of fibroblasts exposed to various stimuli and counterstained for filamentous actin using AlexaFluor594 Phalloidin antibody (red). b Untreated (control) cells exposed to F-gp120 or AF-transferrin or c tamoxifen-treated fibroblasts exposed to F-gp120 (1:200), AF-transferrin (5 μg/ml) or F-Tat (1:50). Fluorescent compounds were added and cells were kept for 30 min on ice and 30 min at 37 °C. Internalization of gp120 and transferrin (green) was visualized by confocal microscope. Scale bar = 10 μm. The experiment was replicated with three independent preparations, in duplicate, with comparable results. Please note that internalization of F-Tat, which is dynamin-independent, occurred even without the presence of dynamins. d) Control fibroblasts were exposed to F-gp120 (1:200, green) or AF-transferrin (5 μg/ml) in the presence of AMD3100 for 30 min at 4 °C and 30 min at 37 °C. AMD3100 pretreatment (500 nM) was initiated 15 min prior to F-gp120 or AF-transferrin application. Images are representative of two independent experiments done in duplicate. Scale Bar = 10 μm